1 Department of Neurobiology and Behavior, 2 Howard Hughes Medical Institute, State University of New York at Stony Brook, Stony Brook, New York 11794; and 3 Department of Physiology, University of Colorado School of Medicine, Denver, Colorado 80262
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ABSTRACT |
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Fully grown oocytes of
Xenopus laevis undergo resumption of the meiotic
cycle when treated with the steroid hormone progesterone. Previous
studies have shown that meiotic maturation results in profound
downregulation of specific endogenous membrane proteins in oocytes. To
determine whether the maturation impacts the functional properties of
exogenously expressed membrane proteins, we used cut-open recordings
from Xenopus oocytes expressing several types of
Na+ and K+ channels. Treatment of oocytes with
progesterone resulted in a downregulation of heterologously expressed
Na+ and K+ channels without a change in the
kinetics of the currents. The time course of progesterone-induced ion
channel inhibition was concentration dependent. Complete elimination of
Na+ currents temporally coincided with development of
germinal vesicle breakdown, while elimination of K+
currents was delayed by ~2 h. Coexpression of human
1-subunit with rat skeletal muscle
-subunit in
Xenopus oocytes did not prevent progesterone-induced
downregulation of Na+ channels. Addition of 8-bromo-cAMP to
oocytes or injection of heparin before progesterone treatment prevented
the loss of expressed currents. Pharmacological studies suggest that
the inhibitory effects of progesterone on expressed Na+ and
K+ channels occur downstream of the activation of cdc2
kinase. The loss of channels is correlated with a reduction in
Na+ channel immunofluorescence, pointing to a disappearance
of the ion channel-forming proteins from the surface membrane.
sodium channels; potassium channels; maturation; internalization; cdc2 protein kinase
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INTRODUCTION |
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FOR MANY YEARS
Xenopus oocytes have served as an excellent system for
elaborating intricate mechanisms of cell cycle control. In vivo, stage
VI immature oocytes are physiologically arrested in the first meiotic
prophase at the G2/M border and resume meiosis when
gonadotropins stimulate surrounding follicle cells, causing them to
secrete the steroid hormone progesterone (46). The
progesterone binds to surface membrane receptors (8, 43)
and initiates oocyte maturation, a crucial process transforming the
immature oocyte into a fertilizable egg. This stimulation of
progesterone receptors is followed by a decrease in cAMP-dependent
protein kinase activity and activation of a cascade of multiple protein kinases: Mos, Raf, mitogen-activated protein kinase, and activation of
cdc25 phosphatase (13, 20). These events lead to
activation of a universal cytoplasmic maturation promoting factor (MPF)
(39), a heterodimer composed of a regulatory subunit
(cyclin B) and a catalytic subunit (cdc2 protein kinase)
(34). Activated MPF then induces nuclear envelope
disassembly (germinal vesicle breakdown; GVBD), comprising chromosome
condensation accompanied by spindle formation and profound cytoskeletal
reorganization (6, 38). During progesterone-induced
maturation, oocytes undergo remarkable structural reorganization. The
early events include progressive size reduction of the microvilli,
flattening of the plasma membrane, movement of cortical granules away
from the plasma membrane, and a significant decrease in the density of
intramembrane particles. After several hours of progesterone treatment,
changes in transmembrane fluxes of Cl, Na+,
K+, and Ca2+ have been detected as well as
membrane potential depolarization, an increase in resistance, and a
decrease in membrane capacitance (31). A striking
phenomenon of selective endocytosis of native membrane-bound proteins
that accompanies meiotic maturation has been well established in
oocytes (44, 45, 51, 60).
The process of internalization has been implicated in the modulation of voltage-operated Ca2+ channels in rat pituitary cells (36) and human neuroblastoma cells (49) and of Na+ channels in fetal rat brain neurons (17). Such a process has also been reported for agonist-induced regulation of adrenoreceptors (27) and cystic fibrosis transmembrane conductance regulator chloride channel (52).
Our studies utilized the fact that a variety of cloned ion channels can be heterologously expressed in Xenopus oocytes and that their functional properties can be monitored during progesterone-induced maturation. The results presented in this paper indicate that exogenously expressed Na+ and K+ channels undergo complete loss during progesterone-induced maturation. The present study indicates that the progesterone-triggered signal transduction pathway downstream of cdc2 kinase activation is responsible for the channel loss.
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MATERIALS AND METHODS |
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Obtaining and handling Xenopus oocytes.
Sections of ovary were surgically isolated from anesthetized
Xenopus frogs (Nasco, Fort Atkinson, WI). After extensive
washing, the follicle cell layer was mechanically removed from stage
V-VI oocytes without any enzymatic pretreatment. Enzymatic
digestion was avoided because of the possible modification of membrane
proteins by collagenase, usually employed to free oocytes from follicle cells. Cells were allowed to recover overnight in a nutrient OR-3 medium containing 50% L-15 medium, 100 µg/ml gentamicin, 4 mM glutamine, and 30 mM Na-HEPES, all from GIBCO BRL (Grand Island, NY),
with pH adjusted to 7.6 with NaOH. The next day, oocytes were
individually injected with 100-140 ng of RNA coding for one of the
following -subunits: rat skeletal muscle (SkM1; Ref.
65), rat peripheral nerve (rPN1; Ref. 64),
human peripheral nerve (hPN1; Ref. 32; kindly provided by
Dr. F. Hoffman), rat brain type IIa (RBIIa; Ref. 4; kindly
provided by Dr. A. Goldin), rat Kv1.4 (Ref. 54; generous
gift of Dr. Lily Jan), or rat DRK1 (Ref. 21; kindly
provided by Dr. Rolf Joho). In the experiments in which the effect of
human
1-subunit (Ref. 37; kindly provided by Dr. A. George) was tested, the oocytes were injected with a mixture
of 75 ng of
RNA and 25 ng of
1 RNA. The methods used to synthesize the RNA were identical to those previously published (47). Microinjection was performed with a Drummond
Nanoject (Drummond Scientific, Broomall, PA). Injected oocytes were
maintained at 18°C in OR-3 medium for up to 5 days until the desired
expression level was achieved. However, oocytes could be routinely
maintained for at least 2 wk in OR-3 medium without signs of deterioration.
Drugs. Progesterone, heparin, taxol, and phalloidin were obtained from Sigma. Roscovitine, brefeldin A, and Ro 20-1724 were purchased from Biomol. Olomoucine and thapsigargin were obtained from LC Laboratories. Calpain inhibitor was purchased from Calbiochem, and 8-bromo-cAMP (8-Br-cAMP) was from RBI. The stock solutions of progesterone, taxol, roscovitine, olomoucine, brefeldin A, Ro 20-1724, thapsigargin, and calpain inhibitor were prepared in dimethyl sulfoxide (DMSO).
Electrophysiology.
Na+ and K+ currents were measured by using a
standard cut-open voltage-clamp technique (63) within
2-5 days after the RNA injection. Currents were generally recorded
from the animal side of the oocyte at 21-22°C. For the cut-open
oocyte voltage-clamp recordings, the three-compartment chamber provided
with the CA-1 voltage clamp (Dagan, Minneapolis, MN) was used. Both top
and guard chamber solutions contained 110 mM
NaCH3SO3, 2 mM
Ca(CH3SO3)2, and 10 mM Na-HEPES at
pH 7.2. The bottom chamber contiguous with the cell interior contained
a solution composed of 120 mM KCH3SO3, 1 mM
K-EGTA, and 10 mM K-HEPES at pH 7.2. Agar bridges filled with 120 mM
NaCH3SO3 and containing a black platinized
platinum wire were used to pass current and control the chamber
potentials. An intracellular micropipette filled with 3 M KCl (~100
k) measured the membrane potential. Currents were acquired with the
use of a CA-1 oocyte clamp amplifier (Dagan). Oocytes that showed an obvious lack of proper voltage control were discarded.
Immunocytochemistry.
Immunohistochemical staining of Xenopus oocytes was
performed to determine whether progesterone treatment led to the
disappearance of Na+ from the plasma membrane. Twenty
oocytes were injected with 100 ng of SkM1, and an additional ten
oocytes were sham injected with distilled water as a control. After
injection, the oocytes were kept at 18°C for an additional 48 h
to allow for maximal expression of functional Na+ channels.
At this time, 10 of the SkM1 injected oocytes were treated with 20 µM
progesterone for 10 h. All of the oocytes were fixed for 2-4
h at 4°C in 0.1 M phosphate-buffered saline (PBS) at pH 7.3 containing 4% paraformaldehyde. After a 1-h rinse in PBS, the oocytes
were cryoprotected through a series of sucrose phosphate buffers,
embedded in Lipshaw embedding compound, and frozen on dry ice. Sections
were cryostat cut at 8-µm thickness, thaw mounted onto slides, and
stored at 20°C. The sections from sham-injected, SkM1-injected, and
progesterone-treated oocytes were pretreated for 1 h in PBS
containing 4% goat serum, 2% bovine
-globulin, and 0.3% Triton
X-100 (PBS-GBT) at 21°C. After a brief rinse in PBS, the sections
were treated overnight with a 1:100 dilution in PBS-GBT of a primary
antibody directed against a highly conserved Na+ channel
epitope (18). Control sections were preblocked with primary antibody along with a 100 M excess of the peptide antigen for
3 h at 21°C. After three rinses in PBS, the sections were incubated for 1 h in a PBS-GBT-containing 1:100 dilution of goat anti-rabbit secondary antibody conjugated to Alexa 568 (Molecular Probes, Eugene, OR). After three more rinses in PBS, the sections were
air dried and coverslip mounted with Vectashield anti-fade medium
(Vector Laboratories). Sections were viewed with a Zeiss LSM 510, version 2.5, confocal laser scanning microscope by using a helium-neon
laser with a 543-nm emission wavelength. Optical images of 0.4 µm
were collected of each oocyte preparation. Images were processed with
Adobe Photoshop 5.0.
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RESULTS |
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Progesterone treatment leads to the complete suppression of
currents associated with expressed Na+
and K+ channels.
Cut-open oocyte voltage-clamp recordings were used to test the effects
of progesterone treatment on the amplitude of currents associated with
exogenously expressed Na+ and K+ ion channels.
In the cut-open mode of voltage clamp, Na+ and
K+ currents were stable over time (>30 min) without any
noticeable change in amplitude or kinetics. Groups of oocytes were
incubated with progesterone in external solution, and amplitudes of
expressed currents were measured at different times in randomly
selected oocytes during the progesterone application. Incubation of
oocytes with 1 µM progesterone for up to 10 h did not affect
ionic currents, and no signs of maturation were detected (data not
shown). In contrast, treatment with 10 µM progesterone resulted in
almost 50% inhibition of Na+ currents after 6 h and
the appearance of a white spot in 10% of oocytes (data not shown). The
most uniform oocyte response in terms of current suppression and
maturation was detected with 20 µM progesterone incubation. Control
experiments with long-lasting DMSO treatment showed no effect on
current amplitude and did not trigger maturation. The Na+
currents resulting from expression of -subunit of rat skeletal muscle (SkM1) Na+ channels consistently displayed a
reduction of peak amplitude during treatment with 20 µM progesterone
(Fig. 1). On closer examination of the
data, the inhibition was found to be insignificant during the first
4 h of progesterone treatment. However, after 4 h, the progesterone-induced block of the Na+ current became
profound and eventually resulted in a complete suppression.
Additionally, coexpression of the human
1-subunit with
SkM1
-subunit in Xenopus oocytes led to fast-inactivating Na+ currents that also disappeared during treatment with
progesterone (Fig. 2). Almost total
elimination of Na+ currents coincided with the time of GVBD
as judged by the appearance of a white spot in the oocyte animal
hemisphere. Progesterone treatment uniformly diminished Na+
current peak amplitude at all voltages (Fig. 1, B and
C), and inhibition of Na+ currents was not
accompanied by any substantial change in kinetics of inactivation or
voltage dependence of activation of the current (Fig. 1, D
and E). We also observed a similar time course of
progesterone-induced inhibition of Na+ currents resulting
from expression of
-subunit of human peripheral nerve (hPN1), rat
peripheral nerve (rPN1), and rat brain type IIa (RBIIa) Na+
channels.
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Involvement of cAMP in progesterone-induced effects on expressed
ion channels.
In a complex cascade of progesterone-induced early signaling events,
the decrease in oocyte cAMP level is well established and is thought to
be critical in the meiotic resumption process (6, 61). On
binding to its plasma membrane receptors, progesterone leads to a G
protein-mediated sharp decrease in adenylyl cyclase activity
(23). Decline in cAMP levels causes a decrease in protein kinase A activity that, in turn, leads to dephosphorylation of putative
maturation-inhibiting proteins and subsequent activation of MPF. On the
other hand, it is known that cAMP-dependent phosphorylation can also
modulate activity of voltage-gated ion channels (12). To
test whether cAMP mediates the effect of progesterone on expressed Na+ and K+ channels, we incubated oocytes with
2 mM 8-Br-cAMP, a membrane-permeable analog of cAMP. A relatively high
concentration of 8-Br-cAMP was selected in light of previous reports
that Xenopus oocytes have very low permeability to cAMP and
high endogenous activity of cAMP phosphodiesterase (9).
Treatment with 8-Br-cAMP did not show any substantial effect on the
level of Na+ and K+ channel expression but
effectively prevented the progesterone-induced inhibition of expressed
currents (Fig. 5A). Moreover,
progesterone treatment (up to 10 h) did not result in the
appearance of a distinct white spot in the oocyte animal hemisphere, a
sign of GVBD, when 8-Br-cAMP was present in the incubation medium. This
observation suggests that elevated level of cAMP in oocytes inhibits
the development of progesterone-induced maturation and confirms similar
earlier findings (9). In contrast, we were unable to
confirm other reports that inhibitors of cAMP phosphodiesterase block
oocyte maturation (9). Incubation of oocytes with 50 µM
Ro 20-1724, a selective inhibitor of cAMP phosphodiesterase
(53), did not prevent progesterone-induced inhibition of
expressed currents (Fig. 5B) or maturation as scored by the
appearance of an animal hemisphere white spot.
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Involvement of the inositol 1,4,5-trisphosphate-sensitive pathway in progesterone-induced inhibition of expressed channels. Studies have clearly implicated the phospholipid signaling pathway in meiotic resumption of Xenopus oocytes (42). To test whether inositol 1,4,5-trisphosphate (IP3)-stimulated events play a role in progesterone effect on expressed ion channels, we used heparin, a potent antagonist of IP3 receptors (24, 11). Microinjection of 40 nl of 200 µM heparin into oocytes immediately before progesterone treatment inhibited the progesterone-induced effects (Fig. 5C). Microinjection of heparin per se did not induce significant changes in the level of the expressed currents. It is established that intracellular Ca2+ levels are regulated by IP3, and it is likely that IP3-sensitive Ca2+ stores are responsible for the transient increase of free calcium associated with progesterone-induced maturation (24). We found that oocyte incubation with 200 µM thapsigargin, which causes a depletion of IP3-sensitive Ca2+ depots by inhibiting Ca2+-ATPase responsible for maintaining high Ca2+ concentration in intracellular organelles (28), did not induce maturation or inhibition of expressed channels (Fig. 5D).
Free intracellular Ca2+ can activate an array of Ca2+-binding proteins. Among them are Ca2+-regulated proteases that are thought to play a key role in the regulation of the cell cycle. Specifically, the Ca2+-activated cysteine protease calpain was shown to be involved in the control of the meiotic cycle in Xenopus oocytes (57, 58, 70). Incubation of oocytes with 100 µM calpain inhibitor did not affect the time course of progesterone-induced maturation as well as inhibition of expressed channels (data not shown).Disruption of cytoskeleton or cytoplasmic protein traffic is not
involved in progesterone-induced downregulation of expressed ion
channels.
It is well established that all three major filamentous systems of the
cytoskeleton (i.e., actin filaments, intermediate filaments, and
microtubules) undergo a profound reorganization during meiotic resumption in Xenopus oocytes (6). Moreover, it
is also known that the cytoskeleton is involved in the regulation of
the function of endogenous (22, 67) and expressed
voltage-activated channels (59). It is likely that
cytoskeletal rearrangements associated with maturation underlie the
progesterone-induced inhibition of expressed ion channels. To test the
potential role of the cytoskeleton, we incubated oocytes with 100 µM
taxol or 100 µM phalloidin for 30 min before and during progesterone
treatment. Taxol promotes the formation of highly stable microtubules
that resist depolymerization (25, 29), and phalloidin is
an F-actin stabilizer (15, 26). We found that neither
taxol (Fig. 6A) nor phalloidin
affected the progesterone-induced maturation and associated inhibition of expressed channels.
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Involvement of the cdc2 kinase in progesterone-induced inhibition of expressed ion channel. Progesterone triggers the transduction process that culminates in activation of cyclin-dependent protein kinase cdc2. It is well established that this event is universal and common in all eukaryotic cells (48). To clarify the role of cdc2 kinase in progesterone-induced inhibition of expressed ion channels, we took advantage of the recent discovery of several specific inhibitors of cyclin-dependent kinases (1, 40). We used olomoucine and roscovitine, two highly specific inhibitors of cdc2/cyclin B kinase. Oocytes were incubated with 100 µM olomoucine or 100 µM roscovitine overnight before and during progesterone treatment. Both compounds showed no substantial effect on the level of Na+ channel expression but were highly effective in preventing progesterone-induced inhibition of expressed channels (Fig. 6, C and D). Furthermore, progesterone treatment did not result in the appearance of a white maturation spot in the oocytes incubated with olomoucine or roscovitine in contrast to the control group of oocytes (data not shown).
Progesterone-induced inhibition of expressed currents is due to a
decrease in surface membrane expression of ion channels.
The time course of progesterone-triggered downregulation of exogenously
expressed currents suggests that channels may be lost. To address this
issue, we assayed Na+ channels in oocyte surface membranes
by using the antibodies raised to an epitope in the internal loop
linking domains III and IV of pore-forming -subunit
(18). Immunocytochemical experiments were carried out as
described in MATERIALS AND METHODS, and the results are
shown in Fig. 7. We found Na+
channel immunofluorescence clearly present in the plasma membrane of
oocytes injected with SkM1
-subunit (Fig. 7B), while no
Na+ channel-related staining was seen in the surface
membrane region of progesterone-treated oocytes (Fig. 7C).
Thus these results lead to the conclusion that progesterone-induced
downregulation of Na+ currents could be attributed to a
reduction of cell surface expression of channel-forming proteins.
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DISCUSSION |
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Gonadal steroids exert profound effects on both the central nervous system and the peripheral nervous system. It has been accepted that steroid hormones produced by peripheral glands can cross the blood-brain barrier and affect a variety of important brain and spinal cord functions. Moreover, a recent study found that glial cells in the brain and other parts of the nervous system can synthesize steroids de novo from cholesterol (30). In addition to activity in glial cells, neurosteroidogenesis has been established in Purkinje cells, pyramidal neurons in the hippocampus, neurons in the retinal ganglion, and sensory neurons in the dorsal root ganglia (66). There is a growing interest in the effects of steroid hormones at the level of the cell membrane in contrast to their "classic" intracellular role as regulators of transcription (3, 56, 71). Recently, nongenomic steroid action has been widely recognized and has been implicated in altering neuronal excitability (75), synaptic functioning (73), cell membrane ultrastructure, and endoexocytotic activity (3). In this regard, progesterone, in particular, is considered to be one of the most active steroids. Nongenomic electrophysiological effects of progesterone include inhibition of a Ca2+ current in human intestinal smooth muscle cells (7), a broad spectrum of K+ channels in human T lymphocytes (19), several cloned Kv channels expressed in cell lines (19), K+ channels in the plasma membrane of cultured renal epithelioid Madin-Darby canine kidney cells (62), K+ channels in hepatocytes (68), the native voltage-activated tetraethylammonium and 4-aminopyridine-sensitive K+ channels in Bufo oocytes (74), and K+ currents in starfish oocytes (60). In addition, a recent study reports that progesterone treatment leads to selective downregulation of rat eag K+ but has no comparable effect on either Shaker H4, Drosophila eag, or Kv1.4 K+ channels heterologously expressed in Xenopus oocytes (10). In contrast, our results demonstrate that progesterone-induced maturation in Xenopus oocytes is associated with functional inhibition of exogenously expressed SkM1, hPN1, rPN1, and RBIIa Na+ channels as well as Kv1.4 and DRK1 K+ channels. It is possible that faster disappearance of Na+ channels vs. K+ channels is a result of the differential spatial distribution of those channels. Clustering of specific ion channels in the surface membrane is well known and is thought to be based on the differential association of ion channel proteins with cytoskeletal elements.
Previous studies have shown that the Drosophila
K+ channel -subunit homologue Hyperkinetic
(Hk) associates with eag
-subunits when
coexpressed in Xenopus oocytes and protects against a
downregulation in eag current amplitude that was otherwise
observed in response to treatment with progesterone (72).
Furthermore, studies of an activity-induced internalization of the
native Na+ channels in immature brain tissues such as
cultured fetal rat forebrain neurons or early postnatal hippocampal
slices demonstrated that low expression of Na+ channels
-subunit complexes in immature brain neurons correlates appropriately with the ability of Na+ channels to be
internalized (2). To test whether auxiliary subunits would
affect maturation-associated downregulation of exogenous
Na+ channels, we coexpressed human
1-subunit
with
-subunit in Xenopus oocytes. Comparison of the time
courses of progesterone-induced inhibition of Na+ currents
associated with
-subunit alone or
-subunit complexes did not
reveal any substantial differences. Thus functional association of
auxiliary
1-subunit with pore-forming
-subunit is
unable to protect tested Na+ channels from
progesterone-induced downregulation.
Several previous studies demonstrated rapid and reversible inhibition of K+ and Ca2+ channels on progesterone application (7, 19, 62). It was suggested that in some cases progesterone might affect ion channels directly or through membrane-delimited pathways (19). Our study shows that the slow onset of maturation-induced downregulation is incompatible with the direct effect of progesterone on the ion channels and implicates a complex signaling cascade involved in the progesterone-triggered process of meiotic resumption. In a search for second messengers that mediate the observed inhibition of ion channels, we found that preventing progesterone-induced drop in cAMP concentration or blocking IP3 binding would inhibit the maturation process and associated downregulation of expressed channels. It has been generally accepted that progesterone-induced modulations of two well-known signaling pathways, the adenylyl cyclase and phosphatidylinositol pathways, are the initial early steps implicated in meiotic resumption (6, 61). Our observation that currents expressed up to 4 h after the start of progesterone treatment were not affected dramatically leads us to suggestion that early events by themselves contribute little to inhibition of currents. In this respect, early events in progesterone-induced signaling cascade should proceed unobstructed to ensure proper activation of the cytoplasmic MPF and the following functional downregulation of expressed channels. Indeed, we demonstrate that two highly specific inhibitors of protein kinase cdc2, olomoucine and roscovitine, effectively prevent progesterone-induced effects. Therefore, we speculate that biochemical events subsequent to protein kinase cdc2 activation in the pathway of maturation are responsible for downregulation of expressed channels.
There are some apparent contradictions in the implication of Ca2+ in the regulation of meiosis. Some studies report a progesterone-triggered transient increase in intracellular Ca2+ found by using Ca2+-sensitive microelectrodes and the Ca2+-sensitive luminescent protein aequorin (41, 69), while other studies utilizing identical Ca2+-detection techniques found no increase in intracellular free Ca2+ (5, 16, 55). However, despite these obvious inconsistencies, other sufficient evidence exists to indicate that intracellular Ca2+ is important for regulating progesterone-induced maturation (24, 33, 58). In an attempt to test the role of Ca2+ in progesterone-induced effects, we used thapsigargin, which is known to induce a transient increase in intracellular free Ca2+ by depleting IP3-sensitive stores. We found that thapsigargin treatment did not affect functional properties of expressed channels and did not prevent progesterone-induced downregulation of ion channels. Our experiments with thapsigargin confirmed previous reports that an increase in intracellular Ca2+ per se is not sufficient to induce maturation (61). In contrast, heparin effects indicated that IP3-dependent events are obligatory steps in progesterone-induced oocyte maturation. These results together imply that the precise timing of specific steps in a progesterone-triggered sequence of events is very important for successful progression of maturation and the associated downregulation of ion channels.
The antibody labeling experiments have indicated that progesterone
treatment leads to elimination of a pore-forming protein of the SkM1
Na+ channel from oocyte surface membrane. Interestingly,
the retrieval of membrane proteins is known to be associated with
maturation. Previous studies have demonstrated that three distinct
membrane proteins, 1-integrin (45),
U-cadherin (44), and Na+-K+-ATPase
(51), undergo similar internalization during oocyte maturation. This finding points to an evidently common mechanism for
maturation-associated control of membrane proteins. On the basis of the
existing body of evidence, Muller et al. (45) postulated that at least three well-distinguished major events occur in a coordinate way to regulate plasma membrane proteins during maturation: 1) the oocyte plasma membrane is cleared from the proteins
by endocytosis, 2) the insertion of the proteins into the
oocyte plasma membrane is inhibited, and 3) the production
of the membrane proteins increases. Apparently, the observed
disappearance of exogenously expressed Na+ channels is
rather a part of a general regulatory phenomenon that serves to build
up the maternal store of membrane proteins in the egg cytoplasm for
subsequent development. The precise mechanism governing ion channels
retrieval in maturing oocytes remains to be elucidated. Given the great
physiological significance of ion channel modulation, the experimental
approach utilized in this study provides a valuable system to elucidate
molecular determinants governing the excitability of cells during the
cell cycle.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-18205.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Shcherbatko, MRL-San Diego, 505 Coast Blvd. S., Ste. 300, La Jolla, CA 92037 (E-mail: anatoly_shcherbatko{at}merck.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 March 2000; accepted in final form 25 September 2000.
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