62 citations found
J Assist Reprod Genet 13 (7): 573-577 (1996)
A soluble sperm factor gates Ca(2+)-activated K+ channels in human
oocytes.
Dale B, Fortunato A, Monfrecola V, Tosti E
Stazione Zoologica, Villa Comunale, Naples, Italy.
PURPOSE: Our goal was to study the activation current in physiologically
competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa
or aged in vitro, and, in particular, to determine whether a soluble sperm
factor triggers a fertilization current comparable to that observed with
intact spermatozoa and to characterize the current involved. METHODS: The
whole-cell voltage-clamp technique was used on spare metaphase II oocytes,
obtained with patient consent from IVF programs. In this configuration
a soluble fraction from human spermatozoa was microinjected, and the current
recorded. RESULTS: Metaphase II human oocytes generate bell-shaped outward
currents of 400-1000 pA (X = 706 +/- 322; n = 10), following injection
of a cytosolic extract from human spermatozoa. The amount of sperm extract
injected was less than 10% of the total oocyte volume and was equivalent
to 1-10 spermatozoa. A similar current was generated following exposure
to 20 microM of the calcium ionophore A23187 (n = 10). The steady-state
conductance of the oocyte increased from 10 to 19.8 nS (n = 10) following
injection of the sperm factor and from 5.3 to 27.7 nS following ionophore
exposure. Both sperm factor- and ionophore-induced currents were reduced
in amplitude when the unfertilized oocyte was preexposed to 25-75 microM
iberiotoxin (n = 8) and eliminated at a concentration of 100 microM iberiotoxin.
CONCLUSIONS: The data support the hypothesis of a soluble sperm factor
involved in the activation of human oocytes and shows that the initial
activation response in the human oocyte is the gating of Ca(2+)-activated
K+ channels.
Dev Biol 177 (1): 178-189 (1996)
External Mg2+ triggers oscillations and a subsequent sustained level
of intracellular free Ca2+, correlated with changes in membrane conductance
in the oocyte of the prawn Palaemon serratus.
Goudeau M, Goudeau H
Observatoire Oceanographique et de Biologie marine de Roscoff, UPR CNRS,
Roscoff, 9042, France.
We have provided evidence that external Mg2+ induces correlated changes
in [Ca2+]i and membrane current or potential in prawn oocytes without any
requirement of fertilization, using the fluorescent Ca2+ indicator Ca green
dextran and voltage and current clamp methods. Replacement of Mg2+-free
ASW with standard (40 mM Mg2+)ASW triggered a diphasic [Ca2+]i response
consisting of an oscillation period followed by a second state of sustained
[Ca2+]i level devoid of oscillation, lasting about 70 min and up to 3 hr,
respectively. In contrast, oocytes maintained for a long time in Mg2+-free
ASW showed no changes in [Ca2+]i which remained at a basal concentration
of about 0.2 microM. The simultaneous records of [Ca2+]i and membrane or
current changes showed that the oscillation period started with a first
[Ca2+]i peak (about 1.7 microM) and was correlated with an initial transient
membrane hyperpolarization or a transient initial outward current peak.
The first [Ca2+]i peak was followed by a slow decrease in [Ca2+]i followed
by a series of [Ca2+]i transients, concurrent with a slow depolarization
of the membrane or a related inwardly directed current. The oscillation
period ended with an oscillatory plateau of [Ca2+]i (about 0.6 microM)
lasting 49.6 +/- 4.5 min, the onset of which was concomitant with a final
membrane hyperpolarization or a related final outward current. A continuous
contact between external Mg2+ and the oocyte membrane was necessary to
maintain the program of [Ca2+]i and membrane conductance changes. The sources
of Ca2+ mobilization are of internal origin for both the first peak and
the subsequent series of [Ca2+]i oscillations, and are mainly of external
origin for the oscillation plateau and for the second state of sustained
[Ca2+]i level. The first and second step of the cortical reaction occurred
during the oscillatory plateau and the second state of sustained [Ca2+]i
level, respectively.
Dev Biol 174 (2): 322-334 (1996)
Novel postfertilization inward Ca2+ current in ascidian eggs ensuring
a calcium entry throughout meiosis.
Arnoult C, Grunwald D, Villaz M
Laboratoire de Biophysique Mol'eculaire et Cellulaire, CNRS URA 520, Grenoble,
France.
The conductance change after fertilization in the oocyte of the ascidian
Ciona intestinalis has been followed by the whole cell patch-clamp technique.
Two new inward currents, which are absent in unfertilized eggs, are elicited
by hyperpolarization from a holding potential of +20 mV, which is the resting
potential soon after fertilization. These currents reach their maximum
level during the first meiotic division cycle, and then decrease in intensity,
becoming almost undetectable at the 2-cell stage. These currents are most
easily seen at high concentrations of barium. At least one, and likely
both, of these currents appears to be carried by Ca ions. One of the currents
is blocked by low concentrations of gadolinium; the other one is blocked
by higher concentrations, although gadolinium at these levels does not
block fertilization and the associated early depolarizing jump of the eggs.
Thus these currents are not carried by channels that mediate the fertilization
current. However, gadolinium blocks normal transition to 2-cell stage and
blocks current oscillations synchronous to free calcium oscillations that
occur normally in eggs around meiosis II. The electrical signature of calcium-release
activated currents, taken together with these findings, suggests that these
inward currents ensure a calcium entry pathway throughout meiosis. A plausible
function of these currents may be to refill the Ca stores that are depleted
after fertilization and that are required to progress into mitotic cell
division. This interpretation is reinforced by experiments on unfertilized
eggs with intracellular Ca stores depleted by thapsigargin, where both
the newly described currents are observed.
J Gen Physiol 107 (3): 381-397 (1996)
A recombinant inwardly rectifying potassium channel coupled to GTP-binding
proteins.
Chan KW, Langan MN, Sui JL, Kozak JA, Pabon A, Ladias JA, Logothetis
DE
Department of Physiology and Biophysics, City University of New York, New
York 10029, USA.
GTP-binding (G) proteins have been shown to mediate activation of inwardly
rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine
cells. Here, we report functional expression of a recombinant inwardly
rectifying channel which we call KGP (or hpKir3.4), to signify that it
is K+ selective, G-protein-gated and isolated from human pancreas. KGP
expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent)
currents while coexpression with a G-protein-linked receptor, yielded additional
agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain
homolog of GIRK1/Kir3.1) produced much larger basal currents than those
observed with KGP or hGIRK1 alone, and upon coexpression with receptor,
similarly large agonist-induced currents could be obtained. Pertussis toxin
treatment significantly diminished agonist-dependent currents due to either
KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced
basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely
the result of G-protein gating as well. When the two channels were coexpressed
with receptor, the relative increase in current elicited by agonist was
similar whether KGP and hGIRK1 were expressed alone or together. When in
vitro translated or when expressed in Xenopus oocytes or CHO mammalian
cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed
against either KGP or hGIRK1 coprecipitated both proteins coexpressed in
oocytes, providing evidence for the heteromeric assembly of the two channels
and suggesting that the current potentiation seen with coexpression of
the two channel subunits is due to specific interactions between them.
An endogenous oocyte protein similar in size to KGP was also coprecipitated
with hGIRK1.
J Biol Chem 271 (4): 2213-2220 (1996)
Kinetics and specificity of a H+/amino acid transporter from Arabidopsis
thaliana.
Boorer KJ, Frommer WB, Bush DR, Kreman M, Loo DD, Wright EM
Department of Physiology, UCLA School of Medicine 90095-1751, USA.
The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana
was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer
flux, and electron microscopic methods to characterize the biophysical
properties, kinetics, and specificity of the transporter. Uptake of alanine
was H(+)-dependent increasing from 14 pmol/oocyte/h at 0.032 microM H+
to 370 pmol/oocyte/h at 10 microM H+. AAP1 was electrogenic; there was
an amino acid-induced depolarization of the oocyte plasma membrane and
net inward currents through the transporter due to the transport of amino
acids favoring neutral amino acids with shortside chains. The maximal current
(imax) for alanine, proline, glutamine, histidine, and glutamate was voltage
and [H+]o-dependent. Similarly, the imaxH was voltage and [amino acid]o-dependent.
The imax for both H+ and amino acid were dependent on the concentrations
of their respective cosubstrates, suggesting that both ligands bind randomly
to the transporter. The K0.5 of the transporter for amino acids decreased
as [H+]o increased and was lower at negative membrane potentials. The K0.5
for H+ was relatively voltage-independent and decreased as [amino acid]o
increased. This positive cooperativity suggests that the transporter operates
via a simultaneous mechanism. The Hill coefficients n for amino acids and
H+ were > 1, suggesting that the transporter has more than one binding
site for both H+ and amino acid. Freeze-fracture electron microscopy was
used to estimate the number of transporters expressed in the plasma membrane
of oocytes. The density of particles on the protoplasmic face of the plasma
membrane of oocytes expressing AAP1 increased approximately 5-fold above
water-injected controls and corresponded to a turnover number 350 to 800
s-1.
J Membr Biol 147 (2): 207-215 (1995)
A voltage-dependent and pH-sensitive proton current in Rana esculenta
oocytes.
Humez S, Fournier F, Guilbault P
Laboratoire de Physiologie Cellulaire, Universite des Sciences et Technologies
de LILLE, Villeneuve d'Ascq, France.
Voltage clamp technique was used to study macroscopic ionic currents in
Rana esculenta oocytes. Depolarization steps led to the activation of a
single type of outward current (Iout) when contaminant potassium and calcium-dependent
chloride currents were pharmacologically inhibited. The voltage threshold
of Iout activation was 10 mV and this current, which did not inactivate,
presented a deactivation the time constant of 73 +/- 21 msec (n = 26) corresponding
to a membrane voltage of -60 mV. Its reversal potential (Erev) was dependent
on the magnitude of the depolarization and also on pulse duration. These
changes in Erev were thought to reflect intracellular ion depletion occurring
during activation of the remaining outward current. Furthermore, the activation
threshold of Iout was clearly affected by modifications in extracellular
and intracellular H+ concentrations. Indeed, intracellular alkalinization
(evoked by external application of ammonium chloride) or extracellular
acidification induced a rightward shift in the activation threshold while
intracellular acidification (evoked by external application of sodium acetate)
or extracellular alkalinization shifted this threshold toward a more negative
value. Lastly, Iout was dramatically reduced by divalent cations such as
Cd2+, Ni2+ or Zn2+ and was strongly decreased by 4 Aminopyridine (4-AP),
well-known H+ current antagonists already described in many cell types.
Therefore, it was suggested that the outward current was prominently carried
by H+ ions, which may play a key role in the regulation of intracellular
pH and subsequent pH dependent processes in Rana oocyte.
Biophys J 69 (3): 904-908 (1995)
Induction of endogenous channels by high levels of heterologous membrane
proteins in Xenopus oocytes.
Tzounopoulos T, Maylie J, Adelman JP
Vollum Institute, Department of Molecular and Medical Genetics, Oregon
Health Sciences University, Portland 97201, USA.
Xenopus oocytes are widely employed for heterologous expression of cloned
proteins, particularly electrogenic molecules such as ion channels and
transporters. The high levels of expression readily obtained permit detailed
investigations without interference from endogenous conductances. Injection
of min K mRNA into Xenopus oocytes results in expression of voltage-dependent
potassium-selective channels. Recent data show that injections of high
concentrations of min K mRNA also induce a chloride current with very different
biophysical, pharmacological, and regulatory properties from the min K
potassium current. This led to the suggestion that the min K protein acts
as an inducer of endogenous, normally silent oocyte ion channels. We now
report that high levels of heterologous expression of many membrane proteins
in Xenopus oocytes specifically induce this chloride current and a hyperpolarization-activated
cation-selective current. The current is blocked by 4,4'-diisothiocyanostilbene-2-2'-disulphonic
acid and tetraethylammonium, enhanced by clofilium, and is pH-sensitive.
Criteria are presented that distinguish this endogenous current from those
due to heterologous expression of electrogenic proteins in Xenopus oocytes.
Together with structure-function studies, these results support the hypothesis
that the min K protein comprises a potassium-selective channel.
Nature 375 (6532): 599-603 (1995)
An excitatory amino-acid transporter with properties of a ligand-gated
chloride channel.
Fairman WA, Vandenberg RJ, Arriza JL, Kavanaugh MP, Amara SG
Howard Hughes Medical Institute, Oregon Health Sciences University, Portland
97201, USA.
Excitatory amino-acid transporters (EAATs) in the central nervous system
maintain extracellular glutamate concentrations below excitotoxic levels
and may limit the activation of glutamate receptors. Here we report the
cloning of a novel human aspartate/glutamate transporter, EAAT4, which
is expressed predominantly in the cerebellum. The transport activity encoded
by EAAT4 has high apparent affinity for L-aspartate and L-glutamate, and
has a pharmacological profile consistent with previously described cerebellar
transport activities. In Xenopus oocytes expressing EAAT4, L-aspartate
and L-glutamate elicited a current predominantly carried by chloride ions.
This chloride conductance was not blocked by components that block endogenous
oocyte chloride channels. Thus EAAT4 combines the re-uptake of neurotransmitter
with a mechanism for increasing chloride permeability, both of which could
regulate excitatory neurotransmission.
J Physiol (Lond) 484 ( Pt 3): 593-604 (1995)
A monovalent cationic conductance that is blocked by extracellular
divalent cations in Xenopus oocytes.
Arellano RO, Woodward RM, Miledi R
Department of Psychobiology, University of California, Irvine 92717, USA.
1. Native Xenopus oocytes were voltage clamped and exposed to Ringer solutions
containing low concentrations of divalent cations. Oocytes, held at -60
mV, developed a reversible non-inactivating smooth inward current (Ic)
associated with an increase in membrane conductance. 2. Ic was selectively
carried by cations (Na+, K+), indicating that the current was not the result
of a non-specific membrane breakdown, but was due instead to removal of
a blocking effect of divalent cations on a specific population of endogenous
ionic channels located in the oocyte membrane. 3. The blocking effects
of Ca2+ and Mg2+ were voltage dependent, implying action at a binding site
within the pore of the cationic channel. For example, the half-maximal
inhibition (IC50) of Ic by Ca2+ was 61 microM in oocytes held at -60 mV
and 212 microM in oocytes held at 0 mV. 4. The Ic channels could be unblocked
by depolarization of the membrane even in the presence of physiological
concentrations of Ca2+ or Mg2+. The unblocking of the channels was observed
as a slowly developing outward current. 5. The novel cationic current was
substantially reduced following in vitro maturation of oocytes by treatment
with progesterone (10 microM, 4-5 h). 6. The physiological role of Ic channels
remains to be elucidated. Nonetheless, their characteristics explain the
ionic basis of the sensitivity of oocytes to reductions in extracellular
divalent cations and raise the possibility that the channels play a role
in calcium homeostasis.
Proc R Soc Lond B Biol Sci 258 (1353): 229-235 (1994)
Osmo-dependent Cl- currents activated by cyclic AMP in follicle-enclosed
Xenopus oocytes.
Arellano RO, Miledi R
Department of Psychobiology, University of California, Irvine 92717.
The role of adenosine 3',5'-cyclic monophosphate (cAMP) in generating the
osmo-dependent slow inward membrane currents (S(in)) elicited by activation
of follicle stimulating hormone (FSH) or acetylcholine (ACh) receptors
was studied in voltage-clamped, follicle-enclosed oocytes of Xenopus laevis
(follicles). Forskolin (FSK) also generated S(in) currents, and in low
concentrations it potentiated the S(in) currents elicited by FSH but not
those elicited by ACh. Moreover, intra-oocyte injections of cAMP elicited
similar slow inward currents (cAMP-S(in)) that: (i) were carried mainly
by chloride ions; (ii) were abolished by defolliculating the oocytes; and
(iii) were dependent on the osmolarity of the external medium. Compared
with the Ca(2+)-dependent chloride channels that are located in the oocyte
membrane; the cAMP-activated S(in) channels were less permeable to I- and
Br-, and their current-voltage relation did not rectify strongly at negative
potentials. Generation of cAMP-S(in) desensitized the FSH-S(in) currents,
but did not have effects on both the S(in) and the fast chloride current
(F(in)) specifically elicited by ACh. Furthermore, follicular phospholipase
C activation through stimulation of angiotensin II (AII) receptors failed
to generate the current responses elicited by ACh. We conclude that cAMP
acts as a potent second messenger in generating the osmo-dependent Cl-
currents elicited by FSH but not those elicited by ACh. The mechanisms
underlying the ACh responses remain unknown. The osmo-dependent chloride
channels activated by cAMP may play a role in the control of volume of
the follicular cells-oocyte complex.
Am J Physiol 267 (6 Pt 1): C1717-C1722 (1994)
Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+
antiporter in Xenopus oocytes.
Rezai K, Kulisz A, Wasserman WJ
Department of Biology, Loyola University of Chicago, Illinois 60626.
Progesterone-stimulated Xenopus laevis oocytes undergo an increase in their
intracellular pH from 7.3 to 7.7 because of the activation of Na+/H+ antiporters
in their plasma membrane. Activation of Na+/H+ exchangers (NHE) in other
cell systems appears to be regulated by phosphorylation of the NHE protein.
In the current study we demonstrated that cytoplasm taken from steroid-stimulated
oocytes rapidly induced an increase in intracellular pH when microinjected
into full-grown stage VI recipient oocytes. The protein within the cytoplasm
that appears to be responsible for this activity is c-mos kinase. Microinjected
pure mosxe kinase protein rapidly activated the Na+/H+ exchangers in full-grown
recipient oocytes. Furthermore, injected mosxe protein rapidly activated
the Na+/H+ exchangers in smaller progesterone-insensitive stage IV oocytes.
Therefore, it appears that the protooncogene product, p39 c-mos kinase,
which is normally synthesized in full-grown stage VI oocytes in response
to progesterone stimulation, is involved in the upregulation of the Na+/H+
antiporters during oocyte meiotic maturation.
Dev Biol 166 (1): 1-10 (1994)
Cell cycle-related fluctuations in oocyte surface area of the ascidian
Ciona intestinalis after meiosis resumption.
Arnoult C, Georges D, Villaz M
Laboratoire de Biophysique Moleculaire et Cellulaire, CNRS URA 520, DBMS/CENG,
Grenoble, France.
Variations in capacitance or cell surface area were recorded on patch-clamped
eggs of the ascidian Ciona intestinalis between the resumption of meiosis
and the first mitotic cleavages. The membrane surface area increased within
the first minutes after fertilization and then oscillated in phase with
the cell cycles of the two meiotic divisions and first mitotic cleavage.
With drugs, we generated two opposite situations (removal and insertion)
or artificial variation in capacitance. In unfertilized eggs, cytochalasin
induced a drop in capacitance linked to a decrease in calcium current intensity
and specifically disturbed membrane removal linked to the first meiotic
division cycle. It left unaffected the following cycles, in agreement with
previous results that only the first meiosis cycle is microfilament dependent.
In fertilized eggs, membrane removal at each cycle was hindered by emetine,
an inhibitor of protein synthesis. The resulting membrane extrusion was
observed in sections by electron microscopy and was linked to an increase
in calcium current intensity. These fluctuations in surface area never
involved the microtubule network, since nocodazole had no effect on any
cycle. The fluctuations of membrane surface area after meiosis resumption
in phase with cell cycles in Ciona oocytes paralleled the pattern previously
described in the ascidian Boltenia villosa. This may reflect the mechanism
by which the oocyte regulates, with possibly different mediators at each
cycle, the connection between cell surface and internal membrane networks.
This interrelation includes the insertion and removal of ion channels necessary
to developmental control.
Biochem Biophys Res Commun 204 (2): 505-511 (1994)
Protein synthesis is required for the generation of oscillatory current
responses in Xenopus oocyte.
Nakamachi Y, Ando H
Department of Physiology, Kobe University School of Medicine, Japan.
In Xenopus oocyte, direct activation of G-proteins by AIF4- or injection
of inositol 1, 4, 5-trisphosphate evoked Ca(2+)-gated Cl-current responses.
The current responses were smooth and not oscillatory when tested immediately
after the oocyte was isolated. After hours of incubation, the response
became oscillatory. This change was prevented by cycloheximide, a protein
synthesis inhibitor. Results indicate that the characteristics of Ca2+
signaling system have changed from not oscillatory to oscillatory with
time and protein synthesis was required for this change.
Dev Biol 162 (1): 111-122 (1994)
Comparison of defolliculated oocytes and intact follicles of the cockroach
using the vibrating probe to record steady currents.
Anderson M, Bowdan E, Kunkel JG
Department of Biological Sciences, Smith College, Northampton, Massachusetts
01063.
Follicle cells were removed by dissection from early vitellogenic oocytes
of the cockroach Blattella germanica. The vibrating probe was used to record
steady currents from 19 defolliculated oocytes and 19 intact follicles
of the same developmental stage. Defolliculated oocytes generated currents
that were stable and distinguishable (by intensity or selective direction)
from background reference values. Distributions of the intensities of reference
values and experimental values were, in general, similar in both intact
and defolliculated preparations. The patterns of currents generated by
preparations recorded in the mid-sagittal plane were analyzed for both
defolliculated oocytes (n = 8) and intact follicles (n = 10). The larger,
generally more mature preparations in both groups generated patterns of
current similar to the pattern seen in mid-vitellogenic follicles (focused
inward near the germinal vesicle (GV), the presumptive ventral side, and
broadly outward on the apo-GV side, the presumptive dorsal side). Smaller
sized preparations in both groups showed inward or outward current on the
apo-GV aspect and, typically, inward current at the GV. Only two defolliculated
oocytes, and no intact follicles, appeared to generate outward current
at the GV, and we believe this observation resulted from recording slightly
outside the mid-sagittal plane. We conclude that preparations during early-vitellogenesis
initially generate currents without an asymmetric pattern and that the
inward flux at the GV is the first step in developing patterns of currents.
The results suggest that the oocyte (and not the follicle cell epithelium)
is responsible for generating the various patterns of currents observed
in early-vitellogenic stages. At the end of early-vitellogenesis, the follicle
cell epithelium begins to adhere tightly to the oocyte. The possibility
is considered that the follicle cells may influence the currents generated
during mid-vitellogenesis.
J Gen Physiol 103 (2): 217-230 (1994)
Hyperpolarization-activated chloride currents in Xenopus oocytes.
Kowdley GC, Ackerman SJ, John JE 3rd, Jones LR, Moorman JR
Department of Internal Medicine (Cardiovascular Division), University of
Virginia Health Sciences Center, Charlottesville 22908.
During hyperpolarizing pulses, defolliculated Xenopus oocytes have time-
and voltage-dependent inward chloride currents. The currents vary greatly
in amplitude from batch to batch; activate slowly and, in general, do not
decay; have a selectivity sequence of I- > NO3- > Br- > Cl- > propionate
> acetate; are insensitive to Ca2+ and pH; are blocked by Ba2+ and some
chloride channel blockers; and have a gating valence of approximately 1.3
charges. In contrast to hyperpolarization-activated chloride currents induced
after expression of phospholemman (Palmer, C. J., B. T. Scott, and L. R.
Jones. 1991. Journal of Biological Chemistry. 266:11126; Moorman, J. R.,
C. J. Palmer, J. E. John, J. E. Durieux, and L. R. Jones. 1992. 267:14551),
these endogenous currents are smaller; have a different pharmacologic profile;
have a lower threshold for activation and lower voltage-sensitivity of
activation; have different activation kinetics; and are insensitive to
pH. Nonetheless, the endogenous and expressed current share striking similarities.
Recordings of macroscopic oocyte currents may be inadequate to determine
whether phospholemman is itself an ion channel and not a channel-modulating
molecule.
FASEB J 8 (2): 231-236 (1994)
ATP-dependent ionic permeability on nuclear envelope in in situ nuclei
of Xenopus oocytes.
Mazzanti M, Innocenti B, Rigatelli M
Dipartimento di Fisiologia e Biochemica Generali, Universita Degli Studi
di Milano, Italy.
The nuclear envelope represents a structural and functional barrier between
cytoplasm and nucleoplasm. Small molecules and solutes passively cross
the nuclear envelope, whereas the transport of large proteins and RNA requires
metabolic energy. Using in situ Xenopus oocyte nuclei, we characterized
ATP-dependent ionic permeabilities on the external surface of the envelope.
The presence, but not necessarily the hydrolysis, of ATP is crucial to
maintaining the channels in an open state. Localization of the ionic channels
is still unclear. From morphologic and current kinetics data, we suggest
a relation between the ionic channels and the nuclear pores. We try, in
this way, to explain the apparent contradiction between the presence of
ion-selective channels in parallel with large aqueous pores on the nuclear
envelope. Under this hypothesis, variations in the metabolic energy content
of the cytoplasm would induce nucleocytoplasmic passive exchanges. The
distribution and movement of charged particles across the nuclear envelope
may influence many cytoplasmic functions. Regulation of the current by
ATP could play an important role in hormonal stimulation, divalent ion
permeation into the nucleus, and cell cycle mechanisms.
Arch Insect Biochem Physiol 25 (1): 9-20 (1994)
Steady-state gradient in calcium ion activity across the intercellular
bridges connecting oocytes and nurse cells in Hyalophora cecropia.
Woodruff RI, Telfer WH
Department of Biology, West Chester University, PA 19383.
Intracellular activities of K+, H+, Mg2+, Ca2+, and Cl-, measured with
ion selective microelectrodes in the oocyte and the nurse cells in ovarian
follicles of Hyalophora cecropia, indicated that a Ca2+ current is a key
component of the electrical potential that is maintained across the intercellular
bridges connecting these two cells. In vitellogenic follicles, Ca2+ activity
averaged 650 nM in the oocyte and 190 nM in the nurse cells, whereas activities
of the other ions studied differed between these cells by no more than
6%. Incubation in 200 microM ammonium vanadate caused a reversal of electrical
potential from 8.3 mV, nurse cell negative, to 3.0 mV, oocyte negative,
and at the same time the Ca2+ gradient was reversed: activities rose to
an average 3.0 microM in the nurse cells and 1.6 microM in the oocyte,
whereas transbridge ratios of the other cations remained at 0-3%. In immature
follicles that had not yet initiated their transbridge potentials, Ca2+
activities averaged approximately 2 microM in both oocyte and nurse cells.
The results suggest that vitellogenic follicles possess a vanadate-sensitive
Ca2+ extrusion mechanism that is more powerful in the nurse cells than
in the oocyte.
J Physiol (Lond) 468: 275-295 (1993)
Inositol trisphosphate-mediated Ca2+ influx into Xenopus oocytes triggers
Ca2+ liberation from intracellular stores.
Yao Y, Parker I
Department of Psychobiology, University of California Irvine 92717.
1. Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger
by liberating Ca2+ from intracellular stores and by promoting influx of
extracellular Ca2+. We examined whether Ca2+ influx modulates intracellular
Ca2+ liberation in Xenopus oocytes by fluorescence monitoring of cytosolic
free Ca2+ together with voltage clamp recording of Ca(2+)-activated Cl-
membrane currents. Sustained activation of membrane Ca2+ permeability was
induced by intracellular injections of a non-metabolizable InsP3 analogue,
3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (3-F-InsP3), and Ca2+
influx was controlled by applying step changes in membrane potential to
alter the driving force for Ca2+ entry. 2. Negative-going potential steps
evoked intracellular Ca2+ signals comprising two components; an initial
transient peak followed by a slower rise. The initial transient grew steeply
over a narrow (ca 40 mV) voltage range but then increased little with further
polarization, whereas the second component showed a nearly linear voltage
dependence. 3. The transient Ca2+ signal continued to rise almost unchanged
when Ca2+ influx was interrupted by stepping the potential to more positive
values after brief hyperpolarization. In contrast, Ca2+ levels declined
monotonically when positive-going steps were applied after longer intervals
during the second component of the Ca2+ signal. 4. Large Ca(2+)-dependent
transient inward (T(in)) membrane currents were evoked during the rising
phase of the initial Ca2+ signal, but little current was associated with
the second component of the Ca2+ signal. 5. The T(in) currents evoked by
hyperpolarization were mimicked at fixed clamp potential by re-admitting
Ca2+ to the bathing solution, and by flash photolysis of caged Ca2+ loaded
into the oocyte. 6. T(in) currents were strongly inhibited by prior release
of Ca2+ from InsP3-sensitive intracellular stores, and vice versa. Experiments
with paired hyperpolarizing pulses and paired photorelease of InsP3 showed
that responses to both stimuli recovered with similar time courses. 7.
We conclude that the transient Ca2+ signal and associated T(in) current
evoked by hyperpolarization arise because Ca2+ entering the oocyte triggers
regenerative release of Ca2+ from InsP3-sensitive intracellular stores.
Since membrane currents evoked by liberated Ca2+ were much greater than
those evoked by Ca2+ entry per se, a major function of InsP3-mediated Ca2+
entry may be to modulate the activity of intracellular Ca2+ stores.
J Physiol (Lond) 458: 319-338 (1992)
Potentiation of inositol trisphosphate-induced Ca2+ mobilization in
Xenopus oocytes by cytosolic Ca2+.
Yao Y, Parker I
Department of Psychobiology, University of California, Irvine 92717.
1. The ability of cytosolic Ca2+ ions to modulate inositol 1,4,5-trisphosphate
(Insp3)-induced Ca2+ liberation from intracellular stores was studied in
Xenopus oocytes using light flash photolysis of caged InsP3. Changes in
cytosolic free Ca2+ level were effected by inducing Ca2+ entry through
ionophore and voltage-gated plasma membrane channels and by injection of
Ca2+ through a micropipette. Their effects on Ca2+ liberation were monitored
by video imaging of Fluo-3 fluorescence and by voltage clamp recording
of Ca(2+)-activated membrane Cl- currents. 2. Treatment of oocytes with
the Ca2+ ionophores A23187 and ionomycin caused a transient elevation of
cytosolic Ca2+ level when cells were bathed in Ca(2+)-free solution, which
probably arose because of release of Ca2+ from intracellular stores. 3.
Membrane current and Fluo-3 Ca2+ signals evoked by photoreleased InsP3
in ionophore-treated oocytes were potentiated when the intracellular Ca2+
level was elevated by raising the Ca2+ level in the bathing solution. 4.
Responses to photoreleased InsP3 were similarly potentiated following activation
of Ca2+ entry through voltage-gated Ca2+ channels expressed in the plasma
membrane. 5. Ca(2+)-activated membrane currents evoked by depolarization
developed a delayed 'hump' component during sustained photorelease of InsP3,
probably because Ca2+ ions entering through the membrane channels triggered
liberation of Ca2+ from intracellular stores. 6. Ba2+ and Sr2+ ions were
able to substitute for Ca2+ in potentiating InsP3-mediated Ca2+ liberation.
7. Gradual photorelease of InsP3 by weak photolysis light evoked Ca2+ liberation
that began at particular foci and then propagated throughout, but not beyond
that area of the oocyte exposed to the light. Local elevations of intracellular
Ca2+ produced by microinjection of Ca2+ acted as new foci for the initiation
of Ca2+ liberation by InsP3. 8. In resting oocytes, intracellular injections
of Ca2+ resulted only in localized elevation of intracellular Ca2+, and
did not evoke propagating waves. 9. The results show that cytosolic Ca2+
ions potentiate the ability of InsP3 to liberate Ca2+ from intracellular
stores. This process may be important for the positive feedback mechanism
underlying the generation of Ca2+ spikes and waves, and for interactions
between the InsP3 pathway and Ca2+ ions entering cells through voltage-
and ligand-gated channels.
Arch Biochem Biophys 297 (2): 388-392 (1992)
Phosphatidic acid and lysophosphatidic acid stimulate receptor-regulated
membrane currents in the Xenopus laevis oocyte.
Ferguson JE, Hanley MR
Department of Biological Chemistry, School of Medicine, University of California,
Davis 95616-8635.
External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic
acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus
laevis oocytes. The responses were observed in oocytes from which follicular
cells had been removed, indicating they were intrinsic to the oocyte itself.
The lipid-induced Ca(2+)-dependent chloride currents were observed in the
absence of extracellular calcium, were blocked by intracellular injection
of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic
acid, and could not be elicited by direct intracellular injection of the
active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic
acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that
the lipid activities on oocytes were potent. With repeated or prolonged
administration of either active lipid, responses exhibited desensitization.
These results demonstrate that the Xenopus oocyte expresses endogenous
functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic
acid and thus provides a powerful model for characterization of the pharmacology
and transduction pathways of these responses.
Methods Enzymol 207: 339-345 (1992)
Use of stage II-III Xenopus oocytes to study voltage-dependent ion
channels.
Krafte DS, Lester HA
Stage II-III Xenopus oocytes represent a useful extension of the standard
Xenopus oocyte expression system. The oocytes are smaller; the reduction
in membrane area, and therefore capacitance, leads to a faster settling
time during a voltage clamp step. In addition, there appears to be less
Ca(2+)-activated chloride current; this may render the stage II-III oocyte
a useful system for studying K+ channels, where chloride currents can be
a significant problem. Conversely, though, these early stage oocytes may
not be useful for expression of neurotransmitter receptors coupled to phospholipase
C, for such receptors are often monitored by activation of the Cl-current.
The injections of RNA are technically more difficult in stage II-III oocytes.
This can, however, be overcome with some simple modifications of the injection
apparatus, mainly inclusion of a pump or similar device for actual injections.
Brain Res 553 (1): 27-32 (1991)
Membrane currents elicited by the epileptogenic drug pentylenetetrazole
in the native oocyte of Xenopus laevis.
Madeja M, Musshoff U, Kuhlmann D, Speckmann EJ
Institut fur Physiologie, Munster F.R.G.
The effects of the epileptogenic agent pentylenetetrazole (PTZ) on membrane
currents of native oocytes of Xenopus laevis were studied. PTZ elicits
a response that consists of two inward currents. The first one is interpreted
to be due to a decrease of potassium permeability since: (1) the input
resistance is increased; (2) the equilibrium potential is near that of
potassium; (3) the current is decreased during administration of potassium
channel blocking agents; and (4) the PTZ response can be mimicked by blocking
potassium channels without PTZ application. The second one is interpreted
to be due to an increase of chloride permeability since: (1) the input
resistance is decreased; (2) the equilibrium potential is near that of
chloride; and (3) the response is decreased during administration of chloride
blocking agents. These findings correspond to some extent with those made
in neurons.
Arch Biochem Biophys 283 (1): 135-140 (1990)
Diversity in responses from endogenous and expressed mammalian receptors
which cause chloride ion efflux from ovarian follicles of Xenopus laevis.
McIntosh RP, McIntosh JE
Department of Obstetrics and Gynaecology, Wellington School of Medicine,
University of Otago, New Zealand.
Inositol phosphates are produced in ovarian follicles of Xenopus laevis
on activation of endogenous acetylcholine receptors, which also stimulates
Ca2+ release and efflux of Cl- ions detected electrophysiologically. Inositol
phosphates were not detectable on activation of endogenous angiotensin
II receptors which did, however, stimulate both a dose-dependent Ca2+ efflux
and a depolarizing current very similar in maximum size and other characteristics
to those caused by acetylcholine action. In contrast, activation of exogenous
receptors for angiotensin II expressed by microinjected mRNA extracted
from bovine adrenal did form measurable inositol phosphates. Also, the
endogenous electrophysiological responses to angiotensin II and acetylcholine
desensitize homologously but fail to cross-desensitize (Lacy, McIntosh,
and McIntosh, 1989, Biochem. Biophys. Res. Commun, 159, 658-663). It appears
that endogenous ovarian angiotensin II receptors in Xenopus activate a
different transduction mechanism from endogenous acetylcholine receptors
and expressed mammalian adrenal angiotensin II receptors and/or may be
sited in the electrically connected follicular cells rather than in the
oocyte itself.
Am J Physiol 259 (5 Pt 1): C792-C800 (1990)
Xenopus oocyte K+ current. III. Phorbol esters and pH regulate current
at gap junctions.
Greenfield LJ Jr, Hackett JT, Linden J
Department of Physiology, University of Virginia Health Sciences Center,
Charlottesville 22908.
Xenopus follicles consist of a single large oocyte surrounded by a monolayer
of follicle cells attached to the oocyte by gap junctions. Adenosine 3',5'-cyclic
monophosphate (cAMP) activates an outward K+ current which is completely
abolished if follicle cells are removed or if phorbol esters (which have
been reported to reduce gap junctional conductance) are added. In this
study we show that phorbol esters do not reduce cAMP levels in follicles
and that acid pH, another known stimulus for reducing gap junctional conductance,
mimics the action of phorbol esters to inhibit the cAMP-stimulated K+ current.
We also examined electrical coupling between oocytes of pairs of follicles
placed in physical contact (across 2 oocyte-follicle cell and 1 follicle
cell-follicle cell gap junction). Phorbol esters and acid pH (5.5-6.5)
decreased electrical coupling without eliciting a shunt current, since
slope conductance of current-voltage curves recorded during voltage clamp
was simultaneously decreased. Increasing cAMP, which has been reported
to enhance gap junctional conductance in mammalian cells, increased slope
conductance without decreasing electrical coupling between pairs of follicles.
The data suggest that cAMP increases and phorbol esters and acid pH decrease
K+ currents at least in part by effects on gap junctions. The effects of
phorbol esters and acid pH to reduce electrical coupling between oocytes
cannot be due to blockade of K+ channels, since such an action would increase
electrical coupling (as verified by computer simulations). These findings
are consistent with the idea that cAMP-activated K+ currents originate
in follicle cells and are communicated to the oocyte via gap junctions.
Biochem Biophys Res Commun 172 (1): 229-236 (1990)
The effect of myo-inositol 1,4,5-trisphosphorothioate on Cl- current
pattern and intracellular Ca2+ in the Xenopus laevis oocyte.
Ferguson JE, Potter B, Nuccitelli R
Department of Zoology, University of California, Davis 95616.
Microinjection of myo-inositol 1,4,5-trisphosphate into voltage-clamped
Xenopus laevis oocytes or the stimulation of the phosphatidylinositol cycle
elicits a complex Ca2(+)-dependent Cl- current pattern. Microinjection
of myo-inositol 1,3,4,5-tetrakisphosphate causes an immediate release of
Ca2+, but elicits a different Cl- current pattern than myo-inositol 1,4,5-trisphosphate.
We have studied the effects of myo-inositol 1,4,5-trisphosphorothioate,
which can not be converted to myo-inositol 1,3,4,5-tetrakisphosphate. Myo-inositol
1,4,5-trisphosphorothioate caused an immediate release of intracellular
Ca2+, as measured by fura-2 imaging. Myo-inositol 1,4,5-trisphosphorothioate
generated a Cl- current pattern similar to myo-inositol 1,3,4,5-tetrakisphosphate,
not myo-inositol 1,4,5-trisphosphate.
FEBS Lett 267 (1): 22-24 (1990)
Interaction between injected Ca2+ and intracellular Ca2+ stores in
Xenopus oocytes.
Dascal N, Boton R
Department of Physiology and Pharmacology, Sackler Faculty of Medicine,
Tel Aviv University, Ramat Aviv, Israel.
Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300
microns under the membrane), the amplitude of the transient Cl- current
induced by the second injection is several-fold higher than that of the
first one. This 'potentiation' persists even at 60-90 min intervals between
injections. However, in oocytes permeabilized to Ca2+ by the ionophore
A23187 in a Ca2(+)-free solution, the potentiation completely disappears
after 30 min. It is proposed that the injected Ca2+ is largely taken up
by the stores, whereas following the second injection, a higher proportion
of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated
oocytes, the stores lose the accumulated Ca2+ over several minutes and
are then ready to take up Ca2+ again, hindering its arrival at the membrane.
Proc Natl Acad Sci U S A 87 (7): 2813-2817 (1990)
Calcium wave evoked by activation of endogenous or exogenously expressed
receptors in Xenopus oocytes.
Brooker G, Seki T, Croll D, Wahlestedt C
Department of Biochemistry and Molecular Biology, Georgetown University
Medical Center, Washington, DC 20007.
The mRNA encoding the cloned substance K receptor was microinjected into
Xenopus laevis oocytes. After expression of the mRNA, Ca2+ was imaged in
the oocytes with a digital imaging fluorescence microscopy system using
the Ca2(+)-sensitive dyes fura-2 and fluo-3. Application of substance K
caused a dose-related wave of Ca2+ mobilization to spread from a focus
and to elevate the Ca2+ concentration in the oocyte. Activation of endogenous
muscarinic or angiotensin II receptors in noninjected oocytes evoked a
similar response. The Ca2+ rise in oocytes induced by substance K was due
to internal Ca2+ mobilization and was independent of external Ca2+, since
it occurred in Ca2(+)-free medium fortified with 2 mM EGTA. The Ca2+ imaging
was well correlated with ion current measurements of voltage-clamped oocytes.
Imaging, in addition to detecting the spatial spread of Ca2+ across the
cell, was at least as sensitive as voltage clamping and much faster when
screening oocytes for the expression of receptor mRNAs that stimulate Ca2+
mobilization. While it is known that fertilization of Xenopus eggs causes
a spreading wave of Ca2+ mobilization, we found that activation of either
native or newly expressed receptors in oocytes causes a similar change
in Ca2+ distribution.
J Physiol (Lond) 417: 173-195 (1989)
Membrane currents elicited by divalent cations in Xenopus oocytes.
Miledi R, Parker I, Woodward RM
Department of Psychobiology, University of California, Irvine 92717.
1. Membrane currents were recorded from voltage-clamped Xenopus oocytes
in response to bath application of various divalent cations. 2. In oocytes
from 93 of 160 frogs tested, Co2+ ions evoked slow, oscillatory membrane
currents. Sensitivity to Co2+ varied greatly between oocytes from different
frogs, but was relatively consistent for oocytes taken from the same ovary.
Oocytes with high sensitivity had response thresholds of 5-10 microM, and
gave currents greater than 1 microA to 1 mM-CoCl2. In contrast, oocytes
from some frogs gave no oscillatory response even to 10 mM-CoCl2. With
responsive oocytes, Cd2+, Ni2+, Zn2+, Mn2+ and Cr2+ ions (5 microM to 1
mM) also elicited oscillations, whereas Sr2+, Ba2+ and Ca2+ (0.1-10 mM)
showed very little activity, and Mg2+ ions, none. 3. Responses to divalent
cation were well preserved in defolliculated oocytes, indicating they were
generated in the oocyte membrane itself, and were not dependent on the
presence of enveloping follicular cells. 4. The oscillatory currents reversed
around -20 mV (the chloride equilibrium potential) and rectified strongly
at potentials more negative than about -60 mV. The oscillations were mimicked
by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3), were largely
preserved after removal of external Ca2+, but were abolished following
chelation of intracellular Ca2+ by EGTA. Intraoocyte injection of Co2+
ions failed to generate oscillatory currents. 5. Currents elicited by divalent
cations resembled the oocyte's oscillatory responses to acetylcholine and
a serum protein. However, the response to divalent cations was not blocked
by atropine and furthermore, the relative sensitivities to these agonists
varied independently between oocytes from different frogs. 6. We conclude
that extracellular Cd2+, Ni2+, Zn2+, Co2+, Mn2+ and Cr2+ interact with
the oocyte surface to raise cytosolic levels of inositol phosphates. This
causes mobilization of intracellular Ca2+, in turn activating Ca2+-gated
Cl- channels in the oocyte membrane. 7. In addition to the large oscillatory
currents, divalent cations generated small (5-50 nA), smooth, maintained
currents associated with decreases in membrane conductance. The size and
ionic basis of these currents varied between oocytes from different frogs.
8. Zinc ions also elicited smooth currents, associated with an increase
in membrane conductance, and carried predominantly by K+. This response
was specific to Zn2+ and occurred independently of oscillatory Cl- currents.
The K+ current was abolished by defolliculation, was potentiated by the
cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine,and
showed facilitation with K+ currents generated by the adenylate cyclase
activator forskolin.
J Physiol (Lond) 415: 189-210 (1989)
Latencies of membrane currents evoked in Xenopus oocytes by receptor
activation, inositol trisphosphate and calcium.
Miledi R, Parker I
Department of Psychobiology, University of California Irvine 92717.
1. Application of serum to Xenopus oocytes elicits an oscillatory chloride
membrane current, which begins after a latency of several seconds or minutes,
and is mediated through a phosphoinositide-calcium signalling pathway.
We studied the characteristics and origin of this latency in voltage-clamped
oocytes. 2. Bath application of low doses of serum evoked responses beginning
after latencies of 1 min or more. The latency decreased with increasing
dose and reached a minimal value of several seconds that did not decrease
with further increases in serum concentration. Experiments to study this
minimal latency were done by applying brief 'puffs' of serum and other
agonists at high concentrations from a local extracellular pipette. 3.
The mean latency of the response evoked by local serum application was
about 7 s (at 22-24 degrees C), but individual responses showed a wide
variation, from 2 s to over 20 s. Diffusion of serum from the pipette tip
to the membrane did not contribute appreciably to this delay, since short
(less than 100 ms) delays were obtained when KCl was applied in the same
way. 4. Currents evoked by acetylcholine and serotonin, in oocytes induced
to acquire muscarinic and serotonergic receptors following injection of
brain messenger RNA, began following latencies similar to that of the serum
response. 5. The response latency was shorter when serum was applied to
the vegetal rather than the animal hemisphere of the oocyte, even though
smaller currents were obtained. 6. The latency showed a slight dependence
upon membrane potential, becoming shorter with depolarization. 7. Cooling
to temperatures below about 22 degrees C produced a striking lengthening
of the delay, corresponding to a Q10 of about 5. In contrast, above 22
degrees C the temperature dependence was slight, with a Q10 of about 1.25.
8. Intracellular injections of calcium and inositol 1,4,5-trisphosphate
(IP3) evoked chloride currents with short (a few tens of milliseconds)
latency. Short (100 ms) latency responses were also evoked when intracellularly
loaded caged IP3 was photolysed by strong illumination, but weak illumination
gave responses with latencies of over 1 min. 9. Measurements of intracellular
free calcium, made with Fura-2 and Indo-1, showed an increase following
serum application beginning coincident with the onset of the membrane current
response.
Dev Biol 134 (1): 59-71 (1989)
Ionic currents underlying the action potential of Rana pipiens oocytes.
Schlichter LC
Department of Physiology, University of Toronto, Ontario, Canada.
Ionic currents in immature, ovulated Rana pipiens oocytes (metaphase I)
were studied using the voltage-clamp technique. At this stage of maturity
the oocyte can produce action potentials in response to depolarizing current
or as an "off response" to hyperpolarizing current. Reducing external Na+
to 1/10 normal (choline substituted) eliminated the action potentials and
both the negative-slope region and zero-crossing of the I-V relation. Reducing
external Cl- to 1/10 or 1/100 normal (methanesulfonate substituted) lengthened
the action potential. The outward current was reduced and a net inward
current was revealed. By changing external Na+, Cl-, and K+ concentrations
and using blocking agents (SITS, TEA), three voltage- and time-dependent
currents were identified, INa, IK and ICl. The Na+ current activated at
about 0 mV and reversed at very positive values which decreased during
maturation. Inward Na+ current produced the upstroke of the action potential.
During each voltage-clamp step the Na+ current activated slowly (seconds)
and did not inactivate within many minutes. The Na+ current was not blocked
by TTX at micromolar concentrations. The K+ current was present only in
the youngest oocytes. Because IK was superimposed on a large leakage current,
it appeared to reverse at the resting potential. When leakage currents
were subtracted, the reversal potential for IK was more negative than -110
mV in Ringer's solution. IK was outwardly rectifying and strongly activated
above -50 mV. The outward K+ current produced an after hyperpolarization
at the end of each action potential. IK was blocked completely and reversibly
by 20 mM external TEA. The Cl- current activated at about +10 mV and was
outwardly rectifying. ICl was blocked completely and reversibly by 400
microM SITS added to the bathing medium. This current helped repolarize
the membrane following an action potential in the youngest oocytes and
was the only repolarizing current in more mature oocytes that had lost
IK. The total leakage current had an apparently linear I-V relation and
was separated into two components: a Na+ current (IN) and a smaller component
carried by as yet unidentified ions.
J Physiol (Lond) 407: 311-328 (1988)
A study of stretch-activated channels in the membrane of frog oocytes:
interactions with Ca2+ ions.
Taglietti V, Toselli M
Institute of General Physiology, University of Pavia, Italy.
1. We have carried out patch-clamp measurements on a cationic channel in
the plasma membrane of the frog oocyte, which can be specifically activated
by membrane stretch. The kinetics of this channel also display a distinct
dependence upon membrane potential, the probability of the channel being
open increasing with membrane depolarization. 2. When the patch-clamp pipette
filling solution was standard Ringer solution, the single-channel current-voltage
(I-V) relationship was linear, the elementary conductance being 38 pS and
the reversal potential +7 mV, suggesting very poor selectivity of the channel
for the various cations. 3. The I-V relationship was highly non-linear
having a strong inward-going rectification when Ca2+-free solutions were
used to fill the patch pipette. These solutions also resulted in a selective,
inward cationic permeability through the membrane, with K+ being more permeable
than Na+ greater than Li+ greater than Ba2+ greater than Ca2+. 4. Though
permeant through the stretch-activated channel, Ca2+ inhibited in a concentration-dependent
manner the currents carried by other cations. La3+ (0.1 mM) was also an
effective channel blocker. 5. The inward current carried by individual
cations at a given membrane potential increased with increasing external
cation concentration up to a saturating level, this level being maximal
for K+ and minimal for Ca2+. Also the half-saturating concentration was
maximal for K+ and minimal for Ca2+ at all membrane potentials. 6. In the
presence of a constant Ca2+ concentration (50 microM) increasing [K+] did
not change the absolute level at which the current saturated; however the
half-saturating K+ concentration was greatly increased, indicating competitive
inhibition between Ca2+ and K+ for the same site. 7. The data are consistent
with a model based on Eyring rate theory for current conduction through
ionic channels, in which we assume that the ions capable of entering the
channel compete for a binding site that they must first occupy before proceeding
on. The possible energy profile of the stretch-activated channel was defined
by optimizing the model parameters to obtain the best fit of the experimental
data. Ca2+ was found to have a smaller dissociation constant and much longer
occupancy time than Na+ or K+, thus accounting for its lower permeability
and inhibitory effect on current conduction by other cations through the
stretch-activatable channel.
Experientia 44 (8): 657-666 (1988)
Ionic currents in morphogenesis.
Nuccitelli R
Zoology Department, University of California, Davis 95616.
Morphogenetic fields must be generated by mechanisms based on known physical
forces which include gravitational forces, mechanical forces, electrical
forces, or some combination of these. While it is unrealistic to expect
a single force, such as a voltage gradient, to be the sole cause of a morphogenetic
event, spatial and temporal information about the electrical fields and
ion concentration gradients in and around a cell or embryo undergoing morphogenesis
can take us one step further toward understanding the entire morphogenetic
mechanism. This is especially true because one of the handful of identified
morphogens is Ca2+, an ion that will not only generate a current as it
moves, but which is known to directly influence the plasma membrane's permeability
to other ions, leading to other transcellular currents. It would be expected
that movements of this morphogen across the plasma membrane might generate
ionic currents and gradients of both electrical potential and intracellular
concentration. Such ionic currents have been found to be integral components
of the morphogenetic mechanism in some cases and only secondary components
in other cases. My goal in this review is to discuss examples of both of
these levels of involvement that have resulted from investigations conducted
during the past several years, and to point to areas that are ripe for
future investigation. This will include the history and theory of ionic
current measurements, and a discussion of examples in both plant and animal
systems in which ionic currents and intracellular concentration gradients
are integral components of morphogenesis as well as cases in which they
play only a secondary role. By far the strongest cases for a direct role
of ionic currents in morphogenesis is the polarizing fucoid egg where the
current is carried in part by Ca2+ and generates an intracellular concentration
gradient of this ion that orients the outgrowth, and the insect follicle
in which an intracellular voltage gradient is responsible for the polarized
transport from nurse cell to oocyte. However, in most of the systems studied,
the experiments to determine if the observed ionic currents are directly
involved in the morphogenetic mechanism are yet to be done. Our experience
with the fucoid egg and the fungal hypha of Achlya suggest that it is the
change in the intracellular ion concentration resulting from the ionic
current that is critical for morphogenesis.
Proc R Soc Lond B Biol Sci 234 (1274): 45-53 (1988)
Transient potassium current in native Xenopus oocytes.
Parker I, Miledi R
Department of Psychobiology, University of California, Irvine 92715.
Depolarization of follicle-enclosed oocytes of Xenopus laevis obtained
from some donors elicits, in addition to other responses, a fast transient
outward current. After holding the membrane potential at -100 mV this response
begins to be activated by depolarizations to around -30 mV, and increases
progressively as the voltage is raised further. A striking characteristic
is that the current recovers only slowly (several seconds) from inactivation
following a depolarizing pulse. Because of its outward direction and insensitivity
to removal of extracellular chloride or addition of tetrodotoxin, the current
probably arises largely through a flux of potassium ions. The current was
abolished after treatment of oocytes with collagenase to remove enveloping
cells, and although it was blocked by barium and zinc ions, tetraethylammonium
was relatively ineffective. In addition, the potassium current was unaffected
by 5 mM manganese, suggesting that it does not arise as a consequence of
an influx of calcium into the oocyte.
Proc R Soc Lond B Biol Sci 232 (1268): 289-296 (1987)
Tetrodotoxin-sensitive sodium current in native Xenopus oocytes.
Parker I, Miledi R
Department of Psychobiology, University of California, Irvine 92717.
Depolarization of oocytes of Xenopus laevis usually elicits mainly passive
currents, and a calcium-dependent chloride current. However, oocytes obtained
from some donors show, in addition, a transient inward current on depolarization
to potentials beyond ca. -40 mV. This current is abolished by tetrodotoxin
at submicromolar concentrations, and is prolonged by veratrine; thus, it
probably arises through sodium channels of a type similar to those found
in nerve and muscle cells. However, the kinetics of the sodium currents
varied between oocytes from different donors; this result suggests that
genes encoding different sodium channels may be expressed in oocytes from
different donors. The presence of these native channels may complicate
experiments to study the expression of exogenous sodium channels encoded
by foreign messenger RNAs injected into the oocyte.
Neurosci Lett 83 (1-2): 89-94 (1987)
Vasoactive intestinal peptide (VIP) induces a K+ current in the Xenopus
oocyte membrane.
Reale V, Ashford ML, Barnard EA
MRC Molecular Neurobiology Unit, MRC Centre, Cambridge, U.K.
Evidence, based on voltage-clamp and current-clamp recording, is presented
for endogenous receptors for vasoactive intestinal polypeptide (VIP) on
the cell membrane of the Xenopus oocyte. At normal resting potential a
hyperpolarisation is produced by VIP, associated with an increase in K+
conductance. The response is dose-dependent, with a threshold near 10(-9)M
VIP. The effect is potentiated by forskolin. Of several other types of
neuropeptide tested, only avian pancreatic polypeptide gives a response:
this is similar to that of VIP, but this peptide can also potentiate the
response to VIP.
J Physiol (Lond) 393: 619-634 (1987)
Changes in sodium, calcium and potassium currents during early embryonic
development of the ascidian Boltenia villosa.
Block ML, Moody WJ
Department of Zoology, University of Washington, Seattle 98195.
1. The whole-cell variation of the patch clamp was used to study ion channel
properties in the unfertilized oocyte, and in surgically isolated blastomeres
from 2-, 4-, and 8-cell embryos of the ascidian, Boltenia villosa. 2. The
unfertilized oocyte has three major voltage-dependent currents: a transient,
inward Na+ current; a transient, inward Ca2+ current; and an inwardly rectifying
K+ current. 3. The total surface area of the embryo, either measured by
capacitance or calculated from cell diameters, increased about 2.5-fold
between fertilization and the 8-cell stage. 4. The Na+ current almost completely
disappeared from the embryo by the time of first cleavage and was undetectable
in any of the blastomeres at the 8-cell stage. This loss was too large
to be explained by the dilution of channels in the oocyte due to newly
added membrane. 5. Both the Ca2+ current and the inwardly rectifying K+
current were maintained at constant or slightly increased density through
the first three cleavage cycles. This suggests that these channels are
added along with new membrane during these stages. 6. No differences in
mean current densities of blastomeres of different developmental fates
were detected through the 8-cell stage. 7. Continuous recordings in single
egg cells between fertilization and first cleavage, using two-microelectrode
voltage clamp, revealed the increase in capacitance, Ca2+ current amplitude,
and K+ current amplitude, and the loss of Na+ current predicted from the
blastomere studies.
Proc R Soc Lond B Biol Sci 232 (1266): 59-70 (1987)
Injection of inositol 1,3,4,5-tetrakisphosphate into Xenopus oocytes
generates a chloride current dependent upon intracellular calcium.
Parker I, Miledi R
Department of Psychobiology, University of California, Irvine 92717.
Injection of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into voltage-clamped
oocytes of Xenopus laevis elicited an oscillatory chloride membrane current.
This response did not depend upon extracellular calcium, because it could
be produced in calcium-free solution and after addition of cobalt to block
calcium channels in the surface membrane. However, it was abolished after
intracellular loading with the calcium chelating agent EGTA, indicating
a dependence upon intracellular calcium. The mean dose of Ins(1,3,4,5)P4
required to elicit a threshold current was 4 x 10(-14) mol. In comparison,
inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) gave a similar oscillatory
current with doses of about one twentieth as big. Hyperpolarization of
the oocyte membrane during activation by Ins(1,3,4,5)P4 elicited a transient
inward current, as a result of the opening of calcium-dependent chloride
channels subsequent to the entry of external calcium. In some oocytes the
injection of Ins(1,3,4,5)P4 was itself sufficient to allow the generation
of the transient inward current, whereas in others a prior injection of
Ins(1,4,5)P3 was required. We conclude that Ins(1,3,4,5)P4 causes the release
of intracellular calcium from stores in the oocyte, albeit with less potency
than Ins(1,4,5)P3. In addition, Ins(1,3,4,5)P4 activates voltage-sensitive
calcium channels in the surface membrane, via a process that may require
'priming' by Ins(1,4,5)P3.
J Physiol (Lond) 390: 397-413 (1987)
Calcium current and calcium-activated inward current in the oocyte
of the starfish Leptasterias hexactis.
Lansman JB
Department of Physiology, School of Medicine, University of California,
Los Angeles 90024.
1. Inward currents in the immature oocyte of the starfish Leptasterias
hexactis were studied with a two-micro-electrode voltage clamp. Experiments
investigated the role of Ca2+ in the Na+-dependent plateau of the action
potential. 2. Voltage steps more positive than -55 mV produced inward currents
in normal sea water that activated and then decayed to a non-zero level
with a double-exponential time course. Returning the voltage to the resting
potential produced an inward tail current that relaxed slowly to zero with
a time course of seconds. 3. Replacing Na+ with choline abolished the slowly
decaying component as well as the slow tail current which followed the
end of the voltage pulse. This suggested that inward current in Na+-containing
sea water consisted of a rapidly decaying component that flowed through
Ca2+ channels and a more slowly decaying component carried by Na+. 4. Ca2+
current was isolated in Na+-free sea water. Activation followed a sigmoidal
time course that could be described with m2 kinetics. Inactivation during
a maintained depolarization followed first-order kinetics and was voltage
dependent. 5. When Ba2+ was substituted for Ca2+ as the divalent ion charge
carrier, inward currents in Na+-containing sea water decayed along a single-exponential
time course. The absence of a slowly decaying Na+ current in Ba2+-containing
sea water suggested that Na+ current depended on Ca2+ influx. 6. The effects
of altering Ca2+ influx on the time course of Na+ current were investigated.
Na+ current decayed more rapidly as the test pulse potential was made more
positive, while raising [Ca2+]o slowed the decaying phase without altering
its dependence on membrane potential. 7. Tail currents measured after rapidly
stepping the membrane potential back to the resting level consisted of
a fast component associated with the closing of Ca2+ channels and a slow
component that was abolished by removing Na+. 8. The variation of the amplitude
of the slow component of tail current with the duration of the voltage-clamp
pulse indicated that Na+ current is associated with a time-dependent component
of membrane conductance. 9. Possible mechanisms for the slowly decaying
Na+ current are considered. The results are discussed in relation to the
idea that the conductance change to Na+ follows the time course of Ca2+
accumulation and removal from the cytoplasm.
Pflugers Arch 409 (4-5): 521-527 (1987)
Dissociation of acetylcholine- and cyclic GMP-induced currents in Xenopus
oocytes.
Dascal N, Lotan I, Lass Y
In Xenopus follicular oocytes, activation of muscarinic receptors evokes
a slow potassium current (H-response); a similar current is evoked by intracellular
injection of cyclic guanosine 3',5'-monophosphate, cGMP (Dascal et al.
1984). We have tested the hypothesis that cGMP may be the second messenger
that mediates the opening of K channel by acetylcholine (ACh). ACh elevated
the intracellular level of cGMP with a time course similar to that of the
development of the muscarinic H-response; maximal increase in cGMP concentration
above the control was about 0.2 pmole/oocyte. The amount of injected cGMP
that produced a detectable K current ("threshold dose") varied between
0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log
response curve was about 2.5, suggesting involvement of a biochemical process
with a positive cooperativity of at least 3. Higher doses of cGMP evoked,
in addition to the outward current, an irregular, rapidly developing, long-lasting
inward current, that never reached amplitudes comparable to those of ACh-evoked
Cl currents. The K current elicited by cGMP was insensitive to elevation
or depletion of external Ca. It was potentiated by isobutylmethylxanthine
(IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at
the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 microM)
rapidly and completely inhibited the cGMP response. It is concluded, that
most of the results presented in this report contradict the hypothesis
that cGMP is the intracellular mediator of ACh-induced changes in membrane
conductance in the oocytes.
Proc R Soc Lond B Biol Sci 231 (1262): 27-36 (1987)
Inositol trisphosphate activates a voltage-dependent calcium influx
in Xenopus oocytes.
Parker I, Miledi R
Injection of inositol trisphosphate (IP3) into oocytes of Xenopus laevis
induces the appearance of a transient inward (Tin) current on hyperpolarization
of the membrane. This current is carried largely by chloride ions, but
is shown to depend on extracellular calcium, because it is abolished by
removal of calcium in the bathing fluid or by addition of manganese. Recordings
with aequorin as an intracellular calcium indicator show that a calcium
influx is activated by hyperpolarization after intracellular injection
of IP3 as well as after activation of neurotransmitter receptors thought
to mediate a rise in IP3. Furthermore, by substituting barium for calcium
in the bathing solution, inward barium currents can be recorded during
hyperpolarization. We conclude that intracellular IP3 modulates the activity
of a class of calcium channels, so as to allow an influx of calcium on
hyperpolarization. In normal Ringer solution this then leads to the generation
of a chloride current, because of the large numbers of calcium-dependent
chloride channels in the oocyte membrane.
Proc R Soc Lond B Biol Sci 229 (1255): 209-214 (1986)
Distribution of fertilization channels in ascidian oocyte membranes.
DeFelice LJ, Dale B, Talevi R
Fertilization currents are similar in ascidian oocytes and fragments, irrespective
of the size and global origin of the fragments; this result suggests that
fertilization channels are distributed uniformly over the oocyte surface.
Because no correlation exists between peak current and surface area of
the cell, it is probable that the fertilizing spermatozoon does not open
all available precursor fertilization channels, but a fixed number limited
to an area around its point of entry.
Pflugers Arch 407 (5): 534-540 (1986)
Resting membrane potential and inward current properties of mouse ovarian
oocytes and eggs.
Peres A
The electrical properties of the membrane of the ovarian oocyte at the
germinal vesicle (GV) stage and of the ovulated egg of the mouse have been
studied using a two-microelectrode voltage-clamp technique. The stable
resting potential measured with a single electrode was -38.2 +/- 2.8 mV
SE (18 oocytes, 5 animals) and -27.8 +/- 1.4 mV SE (28 eggs, 8 animals)
in a solution containing 20 mM [Ca2+]0. The lower values appear to be strongly
affected by damage due to electrode insertion. However, there was no evidence
of the resting potential being more negative than -40 to -50 mV. Voltage-dependent
inward current could not be activated from a holding potential (Vh) close
to the resting potential. When Vh was set at -90 mV, depolarizing pulses
activated a transient inward current in both oocytes and eggs. The threshold
voltage, peak voltage and inactivation vs potential curve were very similar
in oocytes and eggs. On the other hand, the current amplitude appeared
reduced in ovulated eggs, whilst times to peak and inactivation time constants
in eggs were significantly longer than in oocytes. In oocytes the inward
current was blocked by 10 mM Co2+ and decreased by lowering [Ca2+]0 to
5 mM similarly to the results reported for eggs. It therefore appears that
GV ovarian oocytes possess Ca2+ channels which differ from those present
in eggs mainly with respect to their kinetic properties. The physiological
role of this inward current remains obscure in both preparations since
they are almost completely inactivated at the resting potential.
Proc R Soc Lond B Biol Sci 228 (1252): 307-315 (1986)
Changes in intracellular calcium and in membrane currents evoked by
injection of inositol trisphosphate into Xenopus oocytes.
Parker I, Miledi R
Intracellular calcium was monitored by the use of aequorin in voltage-clamped
oocytes of Xenopus laevis. Injection of inositol trisphosphate (IP3) into
oocytes elicited slowly rising and decaying aequorin/calcium signals and
produced oscillatory chloride membrane currents. These responses did not
depend upon extracellular calcium, since they could be elicited in calcium-free
solution and after addition of cobalt or lanthanum to block calcium channels
in the surface membrane. We conclude that IP3 causes the release of calcium
from intracellular stores in the oocyte. Injections of calcium gave aequorin
and membrane current responses that were more transient than those seen
with IP3.
J Cell Sci 81: 189-206 (1986)
The extracellular electrical current pattern and its variability in
vitellogenic Drosophila follicles.
Bohrmann J, Dorn A, Sander K, Gutzeit H
We determined the extracellular electrical current pattern around Drosophila
follicles at different developmental stages (7-14) with a vibrating probe.
At most stages a characteristic pattern can be recognized: current leaves
near the oocyte end of the follicle and enters at the nurse cells. Only
at late vitellogenic stages was an inward-directed current located at the
posterior pole of many follicles. Most striking was the observed heterogeneity
both in current pattern and in current density between follicles of the
same stage. Different media (changed osmolarity or pH, addition of cytoskeletal
inhibitors or juvenile hormone) were tested for their effects on extrafollicular
currents. The current density was consistently influenced by the osmolarity
of the medium but not by the other parameters tested. Denuded nurse cells
(follicular epithelium locally stripped off) show current influx, while
an accidentally denuded oocyte produced no current. Our results show that
individual follicles may be electrophysiologically different, though their
uniform differentiation during vitellogenesis does not reflect such heterogeneity.
Dev Biol 112 (2): 405-413 (1985)
The development of calcium and potassium currents during oogenesis
in the starfish, Leptasterias hexactis.
Moody WJ
The development of membrane electrical properties of oocytes of the starfish
Leptasterias hexactis during oogenesis was studied using voltage- and current-clamp
techniques. Two voltage-dependent K currents--the fast transient and inwardly
rectifying--are present early in oogenesis, before the rapid growth phase,
and are maintained throughout oogenesis at the same current density and
kinetics. The inward current, which is composed of a Ca current and a slower
Ca-dependent inward sodium current, is also present early in oogenesis,
but at very low current density. Late in oogenesis, after the oocyte has
grown to full size, the inward current increases in amplitude by about
fivefold, and undergoes major changes in kinetics. These changes are closely
associated with the migration of the germinal vesicle to the cell periphery.
The relationship of these events to electrophysiological changes during
subsequent maturation and fertilization of the oocytes is discussed.
Dev Biol 112 (2): 396-404 (1985)
Hormone-induced loss of surface membrane during maturation of starfish
oocytes: differential effects on potassium and calcium channels.
Moody WJ, Bosma MM
Prior to fertilization, starfish oocytes undergo meiotic maturation, triggered
by the hormone 1-methyladenine (1-MA). Maturation involves a variety of
complex biochemical, morphological, and electrical changes, many of which
are similar to those caused by progesterone in vertebrates. Using voltage-clamp
and ultrastructural techniques to study maturation in starfish, we have
discovered a novel process by which 1-MA alters the electrical properties
of the oocyte. The surface area of the oocyte decreases by more than 50%
during the first hour of maturation, due to the elimination of microvilli,
but the calcium and potassium currents present are affected differently
by the loss of membrane. The amplitudes of both the transient K current
("A-current") and the inwardly rectifying K current decrease, following
the time course of the decrease in surface area, while the Ca current amplitude
remains virtually unaffected, and may even increase in some oocytes. The
kinetics of the currents do not change. This selective removal of K channels
results in a larger and more rapidly rising action potential in the mature
egg, which may aid in the fast block to polyspermy. The differential accessibility
of various ion channels to mechanisms of membrane removal and insertion
may play an important role in the development of excitable cells.
Neurosci Lett 54 (2-3): 179-184 (1985)
Acetylcholine-induced currents at plasma membrane of the frog oocyte.
Toselli M, Simoncini L, Taglietti V, Tanzi F
Although with remarkable variability, membrane permeability in Rana oocytes
can be modified by application of acetylcholine. The experiments were carried
out in voltage-clamp conditions. Like in Xenopus, the responses proved
to be related to activation of muscarinic receptors operating membrane
channels probably selective for Cl-. At differences with Xenopus, the net
acetylcholine-induced current showed remarkable deviation from linearity,
displaying outward-going rectification. Application of acetylcholine typically
produced opening of membrane channels, while in late spring, we observed
the opposite effect in several batches of oocytes.
Dev Biol 108 (1): 102-119 (1985)
Patterns of ionic current through Drosophila follicles and eggs.
Overall R, Jaffe LF
Large steady electrical currents traverse Drosophila follicles in vitro
as well as permeabilized eggs. During the period of main follicle growth
(stages 9-11), these currents enter the anterior or nurse cell end of the
follicles. This inward current acts like a sodium ion influx with some
calcium involvement. During the period of chorion formation (stages 12-14),
foci of inward current also appear at the posterior, posterodorsal, and
anterodorsal regions of follicles in vitro. In stage 14, the posterior
in current acts like a chloride ion efflux. In preblastoderm eggs substantial
currents continue to enter their anterior end; while weaker and less frequent
ones enter their posterior end. We present models in which the currents
during follicle growth are driven by the plasma membrane of the oocyte
nurse cell syncitium; the external currents during choriogenesis are driven
by the follicular epithelium; while the currents through the preblastoderm
egg are driven by its plasma membrane. Measurements of pole-to-pole resistances
and voltages across preblastoderm eggs indicate that the transcellular
currents normally maintain a steady extracellular voltage gradient along
the perivitelline space, with the anterior pole kept negative by perhaps
4 or 5 mV. The developmental significance of these currents is discussed.
J Physiol (Lond) 357: 173-183 (1984)
Chloride current induced by injection of calcium into Xenopus oocytes.
Miledi R, Parker I
Membrane currents of Xenopus oocytes were studied with the membrane under
voltage clamp. Intracellular injection of the calcium-chelating agent EGTA
reduced, or abolished, the transient outward chloride current normally
activated by membrane depolarization. Intracellular injection of calcium
ions evoked large membrane currents, which inverted direction close to
the chloride equilibrium potential. Injections of strontium, or barium,
were less effective than calcium, while magnesium was ineffective. Large
chloride currents could be evoked by calcium injections in oocytes which
showed only small or no transient outward currents. The current activated
by calcium injection increased with increasing depolarization up to high
(ca. +60 mV) positive potentials, even though the transient outward current
was suppressed by strong depolarization. The results indicate that the
transient outward current depends upon entry of calcium through voltage-gated
calcium ion channels and show that the oocyte membrane contains numerous
chloride channels which are activated by intracellular calcium. Only a
few of these chloride channels are activated by depolarization.
J Cell Physiol 121 (3): 576-588 (1984)
Maturation involves suppression of voltage-gated currents in the frog
oocyte.
Taglietti V, Tanzi F, Romero R, Simoncini L
Voltage- and time-dependent currents having slow kinetics have been studied
in plasma membranes of immature oocytes of the european frog, Rana esculenta.
IK, corresponding to an outward flow of K+, is activated at potentials
more positive than about -40 mV, and subserves outward rectification; Iir,
corresponding to an outward flow of Cl-, is activated at potentials more
negative than about -80 mV and subserves inward rectification. Such currents
can act as negative feedback mechanisms in the control of membrane potential
in the immature oocyte and limit to a somewhat restricted range its possible
deviations from resting values. Besides IK, membrane depolarizations to
potentials more positive than about +30 mV are capable of activating INa,
corresponding to outflow of Na+. By contrast, the frog mature egg-cell
has a single voltage- and time-dependent current, IM, activated at potentials
more positive than +30 mV, with properties similar to INa. The disappearance
of IK and Iir along with remarkable reduction in leakage lowers impedance
in the egg membrane. It seems reasonable to suggest that the observed changes
in membrane permeability reflect changes which have taken place along the
maturation process and are of importance for successful fertilization.
J Physiol (Lond) 356: 275-289 (1984)
Induction and disappearance of excitability in the oocyte of Xenopus
laevis: a voltage-clamp study.
Baud C, Kado RT
Electrically excitable, sodium-selective channels are induced in the membrane
of the oocytes of Xenopus laevis when it is submitted to prolonged positive
potentials (Kado, Marcher & Ozon, 1979; Baud, Kado & Marcher, 1982).
Under a long positive voltage-clamp step, the membrane current, initially
outward, becomes inward with a sigmoidal time course. The mean time to
half-maximal inward current (t 1/2) is about 18 s at 16 degrees C when
stepping the membrane potential to +55 mV. The rate of channel induction
was very temperature dependent (Q10 about 5). In an Arrhenius plot, the
t 1/2 for induction at temperatures between 5 and 22 degrees C showed a
single slope. The rate of induction was dependent on the membrane potential,
increasing exponentially with positive membrane potential (e-fold for a
20 mV change). When the membrane was maintained at resting potential after
induction, the ability to produce inward currents with short depolarizing
steps slowly disappeared with a t 1/2 of 4 min at 16 degrees C. The temperature
dependence for disappearance was larger than that found for induction (Q10
about 7). The rate of disappearance was not dependent on holding the membrane
potential in the range -30 to -100 mV. Induction proceeded in calcium-free
medium. Cycloheximide, a potent protein synthesis inhibitor had no effect
(100 micrograms/ml) on the induction rate. Isobutylmethylxanthine (IBMX)
or theophyllin (phosphodiesterase inhibitors) applied externally (10(-4)
M) did not affect the induction or disappearance rates. From the present
results, mechanisms such as protein synthesis or a second messenger (such
as calcium or cyclic AMP) do not appear to be involved. During the depolarization
necessary to obtain induction, another conductance was also activated.
It was more slowly established, appeared to be non-saturable and had a
reversal potential between zero and -10 mV. It was found to be very much
reduced at temperatures below about 16 degrees C.
J Physiol (Lond) 352: 243-263 (1984)
A voltage-gated hydrogen ion current in the oocyte membrane of the
axolotl, Ambystoma.
Barish ME, Baud C
Membrane currents in the immature oocyte of the urodele amphibian Ambystoma
were studied using the two-micro-electrode voltage-clamp technique. A current
carried by H ions (IH) constituted the major portion of outward current
activated by depolarizations from the resting voltage (about -60 mV). Net
inward current was not observed at this developmental stage. The reversal
potential for IH measured from tail currents obtained in two step voltage-clamp
experiments shifted by 54 mV per unit change in external pH between pH
6.9 and 8.4. The reversal potential at pH 7.4 was not affected by changes
in external K or Cl concentrations. A small change in reversal potential
was observed with removal of external Na. The amplitude of IH was not affected
by removal of external Ca (Mg or Sr substitution). Ca ionophore A23187
shifted the current-voltage relation towards negative voltages. Activation
of IH did not appear to depend on Ca influx. The instantaneous current-voltage
relation for IH, measured from tail currents at approximately equal internal
and external H ion concentrations, was linear between -40 and +30 mV. The
steady-state conductance-voltage relationship was sigmoidal with membrane
voltage, and, at pH 7.4, was one-half maximum at about +15 mV (V1/2). The
time courses of activation and deactivation were proportional to 1-exp
(-t/tau). A plot of time constant (tau) against voltage was bell-shaped,
with a maximum near V1/2. These results suggested that the activation of
IH is voltage dependent. Increases in the external H ion concentration
shifted the conductance-voltage and time constant-voltage relations in
parallel towards positive voltages. The magnitude of these shifts showed
a lower saturation near pH 9. Low concentrations of external Cd (10-300
microM) reduced current amplitude by shifting the current-voltage relation
in the positive direction. Cd also reduced the limiting slope conductance.
These effects were partially reversible.
J Physiol (Lond) 342: 309-325 (1983)
A transient calcium-dependent chloride current in the immature Xenopus
oocyte.
Barish ME
Ionic currents were studied in immature full-grown Xenopus oocytes using
the two-micro-electrode voltage-clamp technique. Recordings of total membrane
current showed a transient outward peak during depolarizations from the
approximate resting voltage (-70 or -80 mV) to voltages more positive than
-20 mV. The current-voltage relation for peak outward current was U-shaped,
with a maximum at about 0 mV. Replacement of external Cl with methanesulphonate
reversed this transient outward current to a transient inward current.
Current relaxations recorded after the membrane potential was stepped to
different voltages at the time of the peak showed a component that inverted
at about -25 to -30 mV. This value was close to ECl as determined by measurement
of the intracellular Cl ion concentration. The reversal potential for these
current relaxations changed with the external Cl concentration as predicted
by the Nernst relation. Replacement of external Ca with Mg, Sr or Ba, or
addition of low concentrations of Ni in the presence of Ca, eliminated
the transient outward current. Increasing the external Ca concentration
increased the amplitude of the transient outward current without affecting
the amplitude of the steady-state current. It was concluded that the outward
peak in records of total membrane current represented the contribution
of a transient outward current carried by Cl ions which was dependent on
the entry of external Ca. It will be noted as ICl(Ca). Decay of ICl(Ca)
could be described at the normal Ca concentration by a single exponential
function whose time constant showed a shallow U-shaped voltage dependence.
ICl(Ca) was maximally activatable by depolarizations from a holding potential
of about -100 mV, but could not be activated by depolarizations from -40
mV. The amplitude of ICl(Ca) showed a large temperature dependence as compared
to the steady-state current, suggesting complex control of its activation.
Boll Soc Ital Biol Sper 59 (8): 1149-1155 (1983)
Electrophysiological study of the frog egg at various stages of development.
IV. Changes associated with the maturation process
Taglietti V, Tanzi F, Romero R
The I/V relationship of frog oocytes is markedly modified following maturation,
lacking the mature unfertilized egg cell membrane of K-selective channels
and the leakage being strongly reduced. The only kind of voltage-dependent
channels still present in this membrane are activated by depolarizing steps
to potentials more positive than 30-40 mV; the current flowing through
these channels is carried at least partly by Na+. At difference with the
immature oocyte, where several ionic mechanisms are oriented to maintain
the membrane potential at a constant level, the mature egg-cell admits
considerable voltage deflections. The observed variations in membrane properties
could be of importance in allowing the maturation process and/or in preparing
the mature egg-cell to successful fertilization.
Boll Soc Ital Biol Sper 59 (7): 949-954 (1983)
Electrophysiological study of frog eggs at different stages of development.
II) Outward rectification in oocytes at the final stage of vitellogenesis
Taglietti V, Tanzi F, Romero R
Voltage-clamp experiments in full-grown frog oocytes, in a range of membrane
potentials from 90 mV negative to 30 mV positive, have revealed the presence
of voltage-dependent channels selective for K+, blocked by extracellular
TEA. The percentage of open K+-channels increases with membrane depolarizations
over a range from -40 mV to +10 mV, thus supporting the outward rectification
in the I/V relationship. The current transport through the K+-channels
open at different potential levels and in various [K+]o takes place in
accordance with the constant-field assumptions. The leakage current of
the oocyte membrane was found to be considerable large.
Dev Biol 98 (1): 60-69 (1983)
A role for action potentials in maturing Rana pipiens oocytes.
Schlichter LC
Intracellular electrical recording and voltage-clamp techniques were used
to investigate possible roles for the action potentials (ap's) of Rana
pipiens oocytes. The peak of each spontaneous or evoked ap is at or near
the equilibrium potential for Na; therefore, the internal Na+ activity
(aiNa) can be calculated from the Nernst equation. During maturation from
metaphase I to metaphase II, aiNa increases from about 5 to 23 mM. By using
three methods to prevent ap's from firing (voltage clamping, increasing
the mechanical damage, adding 5 mM CoCl2 to the Ringer's) it was shown
that the increase in aiNa required the existence of ap's. External Co2+
appeared to prevent the ap's by blocking only the Na+ current. Calculations
showed that the Na+ influx during an ap could account for the observed
increase in aiNa if a gradient or nonuniform distribution of Na+ exists
within the oocyte. Preventing the ap's also delayed the onset of shock
activatibility, a criterion of maturity. I propose that ap's load the oocyte
with Na+ which may regulate the rate of maturation either directly or indirectly.
Dev Biol 98 (1): 47-59 (1983)
Spontaneous action potentials produced by Na and Cl channels in maturing
Rana pipiens oocytes.
Schlichter LC
The electrical excitability of maturing Rana pipiens oocytes was studied
using intracellular recording and voltage-clamp techniques. Naturally ovulated
oocytes, removed from the body cavity within a few hours after ovulation,
possess voltage-sensitive Na and Cl channels that can produce action potentials
(ap's). Young oocytes (sometime during metaphase I to first polar body
stage) can generate trains of spontaneous action potentials: no chemical
treatment or current injection is required. This is the first report of
spontaneous repetitive firing in an egg cell membrane. As the oocyte matures,
the duration of each ap increases because the outward Cl- current decreases.
Middle-aged oocytes (about first polar body stage to metaphase II) have
continuously positive membrane potentials (Vm's). Mature, activatable (metaphase
II) oocytes have negative Vm's when impaled but can produce a long-lived
ap when depolarizing current is injected. The ap's differ fundamentally
from ap's in other excitable cells, including eggs: the Na+ current develops
slowly and does not inactivate; most of the outward current is carried
by Cl-, not by K+; the Cl channel is lost or is rendered insensitive to
voltage as the oocyte matures.
Proc Natl Acad Sci U S A 80 (10): 3096-3100 (1983)
Developmental regulation of Ca2+ and K+ currents during hormone-induced
maturation of starfish oocytes.
Moody WJ, Lansman JB
Changes in the electrical properties of starfish oocytes during hormone-induced
maturation (the reinitiation of meiosis prior to fertilization) were studied
by using the voltage-clamp technique. Three voltage-dependent ionic currents
dominate the current-voltage relation of the immature oocyte: an inward
Ca2+ current, a fast transient K+ current similar to the "A current" of
molluscan neurons, and an inwardly rectifying K+ current. During in vitro
maturation stimulated by the natural maturing hormone 1-methyladenine,
gradual changes in the amplitudes of all three currents were seen: the
Ca2+ currents became larger, and both K+ currents became smaller. The kinetics
of the currents were not significantly altered during maturation. As a
result of these changes, action potentials in the mature egg had lower
thresholds, faster rates of rise, and larger overshoots than those of the
immature oocyte. We also found that the total membrane capacitance decreased
substantially during maturation, perhaps indicating a decrease in membrane
surface area triggered by the hormone. The significance of these results
is discussed in terms of the preparation of the immature oocyte for fertilization
and the mechanisms of modification of ion channel properties during development.
Pflugers Arch 395 (1): 84-86 (1982)
Na and Ca spikes produced by ions passing through Ca channels in mouse
ovarian oocytes.
Yoshida S
Ovarian oocyte membrane of the mouse was found to be excitable. Ca-dependent
action potentials, which were blocked by Co2+, indicated the existence
of Ca channels. In addition, Na-dependent action potentials were detected
in Ca2+-free solution. These Na spikes were insensitive to tetrodotoxin
(TTX) and were blocked by Co2+, Cd2+, or La3+, suggesting that the Na+
goes through the Ca channel instead of the Na channel. Such a Na current
has not been reported in other eggs. It is concluded that both Na+ and
Ca2+ pass through the Ca channels during excitation in mouse ovarian oocytes.
Nature 298 (5874): 572-574 (1982)
Adenosine-induced slow ionic currents in the Xenopus oocyte.
Lotan I, Dascal N, Cohen S, Lass Y
Adenosine and its 5'-phosphorylated congeners evoke specific membrane-mediated
responses in excitable tissues. Available data suggest that inhibition
of the target cell occurs due to hyperpolarization, and in some preparations
a compound effect of ATP (excitation and inhibition) has been found. However,
the ionic mechanism of the purinergic-mediated response has not been studied
by standard intracellular voltage-clamping techniques. Recently, we have
discovered purinergic receptors in the Xenopus oocyte, a well defined giant
cell amenable to rigorous electrophysiological and biochemical studies.
We report here that in these cells, adenosine-induced slow membrane responses
consisted of an early depolarizing (D) transient current carried by Cl
ions, followed by a steady hyperpolarizing (H) current involving K+ ions.
The relative potency sequence for the D current was ATP congruent to ADP
greater than AMP congruent to adenosine; this order was reversed for the
H current.
Proc R Soc Lond B Biol Sci 215 (1201): 491-497 (1982)
A calcium-dependent transient outward current in Xenopus laevis oocytes.
Miledi R
Membrane currents were investigated in Xenopus laevis oocytes under voltage
clamp. Depolarizing pulses, given from a holding potential of about-100
mV, elicited a transient outward current when the membrane potential was
made more positive than about-20 mV. As the potential was made increasingly
positive the transient outward current first increased and then decreased.
The amplitude of the transient current increased when the external Ca2+
concentration was raised; and the current was abolished by Mn2+. It appears
that when the membrane is depolarized Ca2+ ions enter the oocyte and trigger
an outward current, possibly by opening C1- channels.
Proc Natl Acad Sci U S A 79 (10): 3188-3192 (1982)
Sodium channels induced by depolarization of the Xenopus laevis oocyte.
Baud C, Kado RT, Marcher K
An electrically gated Na+ channel can be made to appear in the membrane
of the Xenopus laevis oocyte by simple depolarization. This membrane normally
responds passively to imposed transmembrane currents with resting potentials
around -60 mV, but when it is held depolarized to more than about +30 mV
it becomes possible to obtain long-lasting regenerative depolarizations
up to +80 mV; these depolarizations can last as long as 20 min. This potential
is due to an "induction" of a Na+-dependent channel that is electrically
gated open and closed. Its threshold for opening is about -20 mV and it
is selective for Na+ over Cs+ and choline+ but is blocked by relatively
small quantities of Li+. When a long voltage clamp step to a positive potential
under ENa (+70 to +90 mV) is applied, an inward current is observed for
many minutes, implying that this channel does not have an inactivation
mechanism. The inward Na+ current is blocked by 0.50 mM tetrodotoxin. When
the membrane is held at or near resting potential, the excitability will
disappear with time, but it can be made to reappear by again depolarizing
the membrane.