An inwardly rectifying swelling- and meiotic cell cycle-regulated anion current carried by the ClC channel splice variant CLH-3b dominates the whole cell conductance of the Caenorhabditis elegans oocyte. Oocytes also express a novel outwardly rectifying anion current termed ICl,OR. We recently identified a worm strain carrying a null allele of the clh-3 gene and utilized oocytes from these animals to characterize ICl,OR biophysical properties. The ICl,OR channel is strongly voltage dependent. Outward rectification is due to voltage-dependent current activation at depolarized voltages and rapid inactivation at voltages more hyperpolarized than approximately +20 mV. Apparent channel open probability is zero at voltages less than +20 mV. The channel has a 4:1 selectivity for Cl− over Na+ and an anion selectivity sequence of SCN− > I− > Br− > Cl− > F−. ICl,OR is relatively insensitive to most conventional anion channel inhibitors including DIDS, 4,4′-dinitrostilbene-2,2′-disulfonic acid, 9-anthracenecarboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid. However, the current is rapidly inhibited by niflumic acid, metal cations including Gd3+, Cd2+, and Zn2+, and bath acidification. The combined biophysical properties of ICl,OR are distinct from those of other anion currents that have been described. During oocyte meiotic maturation, ICl,OR activity is rapidly downregulated, suggesting that the channel may play a role in oocyte Cl− homeostasis, development, cell cycle control, and/or ovulation.
- chloride channel
- cell cycle
- meiotic maturation
anion channels play a variety of essential physiological roles including regulation of cell excitability, transepithelial fluid transport, cell volume control, and acidification of intracellular organelles. Electrophysiological methods have identified innumerable whole cell and single-channel anion currents with diverse biophysical and regulatory properties. These currents define anion channels with conductances of a few to several hundred picosiemens that are regulated by intracellular Ca2+, changes in cell volume, extracellular pH, membrane voltage, second messengers, and neurotransmitters (see, e.g., Refs. 7, 13–15, 17, 24).
Well-established families of anion channel-encoding genes include ligand-gated anion channels (4, 5, 13), ClC channels (14), and CFTR (8). At least some members of the bestrophin gene family encode anion channels (11, 22), and human homologs of the Drosophila gene tweety give rise to anion currents when expressed heterologously (23). Aquaporin 6 may also function as an anion channel in intracellular compartments (12, 25).
The limited number of anion channel-encoding genes that have been identified cannot account for the diversity of anion currents observed by electrophysiological methods. Clearly, additional anion channel-encoding genes must exit. A case in point is the channel(s) that gives rise to the outwardly rectifying swelling-activated anion current ICl,swell. Despite extensive study, the molecular identity of the ICl,swell channel remains unknown and a source of considerable contention (9, 18, 20).
We recently began utilizing the nematode Caenorhabditis elegans for studies of anion channel physiology, molecular biology, and biophysics. C. elegans provides a number of experimental advantages for such studies (1, 21). These advantages include a short life cycle, genetic tractability, and a fully sequenced and well-annotated genome. It is also relatively easy and economical to manipulate and hence characterize gene function in C. elegans.
The worm oocyte is a large and relatively accessible cell type that is particularly well suited for patch-clamp electrophysiology. In wild-type oocytes, the whole cell conductance is dominated by the activity of an inwardly rectifying anion channel, CLH-3b, encoded by the ClC gene clh-3 (6, 19). Oocytes isolated from worms harboring the clh-3 deletion allele ok763 lack CLH-3b currents (6), allowing us to utilize this cell type for physiological and molecular characterization of additional anion channel activity. We demonstrate here that C. elegans oocytes also express a novel outwardly rectifying anion current termed ICl,OR. ICl,OR is strongly voltage dependent and is activated by depolarized voltages. Apparent open probability (Po) of the ICl,OR channel is zero at voltages more negative than approximately +20 mV. During oocyte meiotic maturation, ICl,OR activity is rapidly downregulated, suggesting that the channel may play a role in oocyte ion homeostasis, development, and/or ovulation.
MATERIALS AND METHODS
C. elegans strains.
The worm strain RB900, which carries the clh-3 deletion allele ok763, was used throughout this study. Worms were cultured at 16°C or 20°C with standard methods (2).
Whole cell patch-clamp recording.
C. elegans oocytes were isolated as described previously (19). Unless otherwise noted, experiments were performed on mix-staged, nonmaturing oocytes with cell membrane capacitances between 19 and 34 pF. Patch electrodes were pulled from 1.5-mm outer diameter silanized borosilicate microhematocrit tubes to a tip resistance of 2–4 MΩ. Whole cell currents were measured with an Axopatch 200B (Axon Instruments, Foster City, CA) patch-clamp amplifier. Electrical connections to the patch-clamp amplifier were made with Ag/AgCl wires and 3 M KCl/agar bridges. Data acquisition and analysis were performed with pClampfit 9.2 software (Axon Instruments). The standard bath and pipette solutions contained (mM) 140 N-methyl-d-glucamine (NMDG)-Cl, 2 Ca(gluconate)2, 2 MgSO4, 10 HEPES, and 40 sucrose (pH 7.3, 340 mosmol/kgH2O) and 140 NMDG-Cl, 10 HEPES, and 20 sucrose (pH 7.3, 310 mosmol/kgH2O), respectively. For studies of the pH sensitivity of whole cell currents, the bath solution was buffered with 10 mM 2-(N-morpholino)ethanesulfonic acid.
Data are presented as means ± SE. Statistical significance was determined with Student’s two-tailed t-test for paired or unpaired means. P values of <0.05 were taken to indicate statistical significance.
clh-3(ok763) oocytes express an outwardly rectifying anion conductance.
The ok763 allele is an ∼1,500-bp deletion in the clh-3 gene. Oocytes isolated from wild-type worms express a meiotic cell cycle- and swelling-activated inwardly rectifying anion current carried by the ClC splice variant CLH-3b (6, 19). CLH-3b currents are not detected in oocytes isolated from clh-3(ok763) worms, indicating that ok763 is a clh-3-null allele (6).
The absence of CLH-3b currents, which normally dominate whole cell recordings, enabled us to detect and characterize an outwardly rectifying conductance in clh-3(ok763) oocytes. As shown in Fig. 1A, whole cell currents were evoked by stepping membrane voltage for 1 s between −100 and +100 mV in 20-mV increments from a holding potential of 0 mV. At voltages greater than +60 mV, the current exhibited a time- and voltage-dependent activation (Fig. 1A).
The current was constitutively active on obtaining whole cell access, continued to activate for the first 1–2 min of recording, and then ran down slowly over time (Fig. 1B). Rundown was not prevented by addition of 100 or 420 nM Ca2+, 2 mM ATP plus 2 mM Mg2+, or 2 mM ATP and 0.5 mM GTP plus 2.5 mM Mg2+ to the pipette solution or by complete removal of Ca2+ (data not shown). Cell swelling or shrinkage induced by changes in bath osmolality also had no effect on current amplitude (data not shown).
Figure 1C shows current-voltage (I-V) relationships for the peak whole cell current, and the current after rundown was complete. Surprisingly, in the presence of symmetrical 140 mM NMDG-Cl bath and pipette solutions, the reversal potential (Erev) of the current was strongly hyperpolarized. Mean ± SE Erev values for the peak current and the current observed 12 min after obtaining whole cell access were −19 ± 2 mV (n = 7) and −23 ± 6 mV (n = 5), respectively.
Figure 2A shows an I-V relationship for cells that were patch clamped with bath and pipette solutions in which Cl− was replaced with gluconate. Outward current was dramatically reduced under these conditions. The Erev observed in the presence of symmetrical NMDG-gluconate solutions was similar to that measured in the presence of bath and pipette Cl− (mean ± SE Erev = −27 ± 1 mV; n = 21). Replacement of bath gluconate with Cl− 3–7 min after obtaining whole cell access increased outward current approximately sevenfold at +100 mV. Bath Cl− addition also shifted Erev significantly (P < 0.04) from a mean ± SE value of −24 ± 3 mV to −36 ± 4 mV (n = 5). The increased current amplitude and hyperpolarizing shift in Erev indicate that the outward current is carried by Cl−.
The hyperpolarized Erev of the small current observed in the presence of symmetrical NMDG-gluconate solutions can be explained by either an anion moving into the cell or a cation moving out. The only anion other than gluconate (and OH−) in the bath is SO42− from 2 mM MgSO4. Replacement of SO42− with gluconate failed to depolarize Erev (data not shown), indicating that inward SO42− movement cannot account for the hyperpolarized Erev.
No exogenous outwardly directed cation gradient is present in the NMDG-gluconate bath and pipette solutions. However, it is conceivable that metabolic generation of protons or organic cations produces an outwardly directed cation gradient. Reduction of extracellular pH from 7.3 to 6.3 had no significant (P > 0.6) effect on Erev (mean ± SE Erev at pH 7.3 and 6.3 = −41 ± 4 and −38 ± 5 mV, respectively; n = 4), suggesting that the current and hyperpolarized Erev are due to the efflux from the cell of an unidentified metabolically generated organic cation. We refer to this unidentified current as a “background” current.
Figure 2B shows the mean I-V relationship of whole cell currents observed in the presence of symmetrical NMDG-Cl solutions after subtraction of the mean peak background current. The background-subtracted current reversed at 1.4 ± 3.4 mV (n = 12), which was not significantly (P > 0.7) different from 0 mV. We conclude that clh-3(ok763) oocytes express a strongly outwardly rectifying Cl− conductance, and we term the current carried by the channel ICl,OR. The ICl,OR channel exhibits virtually no inward conductance when whole cell currents are evoked with the voltage step protocol shown in Fig. 1A. The nonzero Erev observed with symmetrical NMDG-Cl solutions reflects the presence of a small inward background current that reverses at hyperpolarized voltages.
Voltage-dependent properties of ICl,OR.
Figure 3A shows a family of ICl,OR traces activated by 1-s depolarizing voltage steps of 0–200 mV. After each voltage step, the current was inactivated by clamping membrane voltage to −80 mV. Figure 3A, inset, shows a family of tail currents recorded during the inactivating voltage step.
The apparent Po of the ICl,OR channel at various test voltages was estimated by normalizing peak tail currents to the peak tail current measured after an activating voltage step of +200 mV. As shown in Fig. 3B, apparent Po is effectively zero at voltages more negative than approximately +20 mV. The half-activation voltage and gating charge for the channel, estimated by fitting a Boltzmann function to the relative tail current data shown in Fig. 3B, were 107 ± 0.5 mV and −1.2 ± 0.03, respectively.
Depolarization-induced activation of ICl,OR at voltages ≥80 mV was well described by the sum of two exponentials that defined fast and slow time constants (τfast and τslow). Both τfast and τslow were strongly voltage dependent and decreased with increasing depolarization (Fig. 3, C and D).
Inactivation of ICl,OR at −80 mV was also described by fast and slow time constants: τfast for inactivation increased approximately twofold over the voltage range of +80 to +200 mV (Fig. 3E), whereas τslow was largely insensitive to activating voltage (Fig. 3F).
To further characterize hyperpolarization-induced inactivation, ICl,OR was activated by 1-s voltage steps to +120 mV and then inactivated at test voltages of −120 mV to −20 mV (Fig. 4A). As shown in Fig. 4, B and C, τfast and τslow were largely insensitive to the hyperpolarizing voltages tested.
Ion selectivity of the ICl,OR channel.
Relative ion permeability (i.e., PX/PCl) of the ICl,OR channel was calculated with the Goldman-Hodgkin-Katz (GHK) equation and measured changes in Erev induced by bath ion substitutions. Because the ICl,OR channel inactivates rapidly at hyperpolarized voltages (Figs. 3 and 4), Erev values could not be measured readily with step voltage-clamp protocols. Instead, ICl,OR was activated by repetitive 1-s steps to +80 mV from a holding potential of 0 mV. After stable current activation was achieved, membrane potential was ramped rapidly from +80 mV to −80 mV at 0.8 V/s (Fig. 5A). As shown in Fig. 5B, significant inward current was detected with this ramp protocol. The mean ± SE Erev of ICl,OR measured with this ramp protocol in the presence of symmetrical 140 mM NMDG-Cl bath and pipette solutions was 0.9 ± 0.7 mV (n = 5). This Erev was not significantly (P > 0.3) different from the predicted value of 0 mV.
In the presence of symmetrical 140 mM NaCl solutions, mean ± SE Erev was 0.6 ± 0.9 mV (n = 9), which was not significantly (P > 0.5) different from the predicted value of 0 mV. Reduction of extracellular NaCl to 70 mM by isosmotic replacement with sucrose depolarized Erev by 10.6 ± 0.4 mV (n = 9). The shift in Erev demonstrates that the ICl,OR channel is permeable to Na+ but is predominantly anion selective. Mean ± SE relative Na+ permeability (PNa/PCl) calculated from the GHK equation was 0.25 ± 0.02 (n = 9).
Relative anion permeability was assessed by complete replacement of bath Cl− with Na+ salts of the test anion. Replacement of bath Cl− with F−, Br−, I−, or SCN− shifted Erev by 19.5 ± 0.03, −8.7 ± 0.02, −13.2 ± 0.04, and −17.8 ± 0.04 mV (n = 5 or 6), respectively. Calculated anion permeability ratios are shown in Fig. 5C. The anion selectivity sequence of the ICl,OR channel is SCN− > I− > Br− > Cl− > F−.
Pharmacology and pH sensitivity of ICl,OR.
ICl,OR was not inhibited by bath addition of 0.5 mM DIDS, 0.5 mM 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), 0.5 mM 9-anthracenecarboxylic acid (9-AC), or 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (data not shown). However, 0.5 mM niflumic acid inhibited ICl,OR (at +80 mV) by 50 ± 3% (n = 6). Exposure to 0.1 mM Gd3+, 10 mM Cd2+, or 10 mM Zn2+ reversibly inhibited ICl,OR (at +80 mV) by 30 ± 1% (n = 6), 78 ± 2% (n = 6) and 83 ± 2% (n = 5), respectively. Figure 6, A and B, show the effect of 4 mM Zn2+ on ICl,OR activity and a Hill plot of Zn2+ inhibition. The mean ± SE Kd and Hill coefficient for Zn2+ inhibition were 1.0 ± 0.3 mM and 0.6 ± 0.1 (n = 5 or 6), respectively. Acidification of the bath to pH 4.8 rapidly and stably inhibited ICl,OR by 87 ± 1% (n = 5; Fig. 6C). Current levels rapidly increased and transiently overshot initial starting levels when bath pH was increased back to pH 7.3 (Fig. 6C).
ICl,OR is also expressed in wild-type oocytes.
To determine whether ICl,OR is present and might play a functional role under normal physiological conditions, we characterized outward anion currents in nonmaturing wild-type oocytes. Figure 7A shows wild-type oocyte whole cell currents evoked by membrane voltages between −100 and +100 mV. Inward currents activated slowly when membrane voltage was hyperpolarized to values more negative than −60 mV (Fig. 7A). As we have described in detail previously (6, 19), this inward current is due to the activity of CLH-3b.
Prominent outward currents were also detected in wild-type oocytes (Fig. 7A). Like ICl,OR, the outward current exhibited time- and voltage-dependent activation at voltages greater than +60 mV. Depolarization-induced activation of this current at +100 mV was well described by the sum of two exponentials that defined τfast and τslow of 14 ± 2 and 641 ± 104 ms (mean ± SE; n = 6), respectively. With the same voltage-clamp protocol (e.g., Figs. 1A and 7A), the fast and slow activation time constants of ICl,OR in clh-3(ok763) oocytes were 14 ± 1 and 509 ± 94 ms (mean ± SE; n = 6), respectively, and were not significantly (P > 0.5) different from those in wild-type oocytes.
The outward current in wild-type oocytes showed a pattern of run-up and rundown that was virtually identical to that of ICl,OR (Fig. 7, B and C; compare to Fig. 1, B and C) and was inhibited 44 ± 8% (n = 3) by addition of 1 mM Zn2+ to the bath. The extent of Zn2+ inhibition was not significantly (P > 0.5) different from that of ICl,OR in clh-3(ok763) oocytes (see Fig. 6B). The outward current was also inhibited by bath acidification (Fig. 7D). Mean ± SE percent inhibition was 86 ± 1% (n = 4), which was not significantly (P > 0.5) different from that observed for ICl,OR. When bath pH was increased back to pH 7.3, current levels rapidly increased and transiently overshot their initial starting levels (Fig. 7D). This rapid recovery and overshoot were also observed in oocytes isolated from clh-3(ok763) worms (see Fig. 6C). On the basis of the results shown in Fig. 7 and discussed above, we conclude that ICl,OR is expressed and functional in wild-type oocytes.
Developmental regulation of ICl,OR activity.
Adult C. elegans hermaphrodites possess two U-shaped gonad arms connected via spermatheca to a common uterus. Oocytes form in the proximal gonad and accumulate in a single-file row of graded developmental stages. Developing oocytes remain in diakinesis of prophase I until they reach the most proximal position in the gonad arm, where they reenter the meiotic cell cycle, a process termed meiotic maturation. Maturing oocytes are ovulated into the spermatheca for fertilization. During development and progression through the gonad, oocyte size increases ∼200-fold before initiation of meiotic maturation and ovulation (10, 16).
As shown in Fig. 8A, oocyte size, which reflects oocyte developmental stage, was inversely correlated with ICl,OR current density. Smaller, younger oocytes exhibited higher current densities compared with larger, more fully developed oocytes. In fully grown oocytes undergoing meiotic maturation, peak current levels were six- to sevenfold lower (P < 0.001) than those observed in oocytes at earlier stages of development (Fig. 8B).
Whole cell current in maturing oocytes underwent a run-up and rundown that was similar to ICl,OR observed in nonmaturing cells (compare Figs. 1B and 7B). However, the voltage-dependent properties of the current in maturing oocytes were considerably different. Instantaneous currents were detected at depolarized voltages, and these currents exhibited a slow linear time-dependent activation (compare Figs. 1A and 7C). In addition, the current was less rectified than that observed in nonmaturing oocytes (compare Figs. 1C and 7D).
We examined the ion selectivity and pharmacological properties of the current in maturing oocytes to assess whether they were carried by the ICl,OR channel. Reduction of extracellular NaCl to 70 mM depolarized Erev by 12.4 ± 0.6 mV (n = 6). The mean ± SE calculated PNa/PCl of the channel was 0.17 ± 0.02. Replacement of extracellular Cl− with F−, Br−, I−, or SCN− shifted Erev by 26.3 ± 1.7, −6.9 ± 0.5, −11.9 ± 0.2, and −19.1 ± 0.7 mV, respectively (n = 5 or 6). Mean ± SE anion PX/PCl for F−, Br−, I−, and SCN− were 0.36 ± 0.03, 1.31 ± 0.03, 1.59 ± 0.01, and 2.11 ± 0.06 (n = 5 or 6), respectively, which yields an anion selectivity sequence of SCN− > I− > Br− > Cl− > F−. Addition of 10 mM Zn2+ or 0.5 mM niflumic acid to the bath inhibited the current by 68 ± 4% (n = 4) and 75 ± 7% (n = 3), respectively. As with the current in nonmaturing oocytes, bath addition of DIDS, 9-AC, or NPPB had no inhibitory effect (data not shown). The cation selectivity, anion selectivity, and pharmacological properties of the outward Cl− currents in maturing oocytes are similar to those in nonmaturing oocytes, indicating that they are carried by the ICl,OR channel.
We also examined whole cell currents in wild-type oocytes undergoing meiotic maturation. ICl,OR in maturing wild-type oocytes showed the same pattern of change, including altered voltage-dependent activation (Fig. 9A) and a six- to sevenfold reduction in amplitude (Fig. 9B). The current also underwent run-up and rundown similar to that observed in clh-3(ok763) oocytes (data not shown).
Our patch-clamp studies on oocytes isolated from wild-type and a clh-3-null mutant worm strain have identified a novel outwardly rectifying anion current, ICl,OR. The strong outward rectification of ICl,OR is due to voltage-dependent current activation at depolarized voltages and rapid inactivation at voltages more hyperpolarized than approximately +20 mV (Figs. 3 and 4). The ICl,OR channel has a 4:1 selectivity for anions over cations and an anion selectivity sequence of SCN− > I− > Br− > Cl− > F− (Fig. 5). ICl,OR is relatively insensitive to most conventional anion channel inhibitors including DIDS, DNDS, 9-AC, and NPPB. However, the current is rapidly inhibited by niflumic acid and metal cations including Gd3+, Cd2+, and Zn2+ (see e.g., Fig. 6, A and B). Bath acidification also inhibits the current (Fig. 6C).
The combined biophysical properties of ICl,OR are distinct from those of other anion currents that have been described. However, two recently characterized anion currents, ICl,mec and ICl,acid, bear some similarity to ICl,OR. ICl,acid is a strongly outwardly rectifying anion current identified in HEK293 cells (15). The ICl,acid and ICl,OR channels have similar voltage-dependent properties and anion selectivities but differ sharply in their pharmacology. In addition, ICl,acid is activated by protons, whereas protons inhibit ICl,OR.
ICl,mec is a strongly outwardly rectifying anion current present in developing C. elegans embryo cells that is activated by membrane stretch (3). The ICl,mec and ICl,OR channels have similar voltage-dependent properties and nearly identical relative cation and anion permeabilities. However, ICl,mec is inhibited by both DIDS and NPPB, and membrane stretch induced by oocyte swelling or shrinkage had no consistent effect on ICl,OR activity.
The molecular identity of the ICl,OR channel is uncertain at present. None of the identified anion channel genes encodes channels with functional properties that recapitulate those of ICl,OR. Some ClC genes give rise to strongly outwardly rectifying currents (14), but the ICl,OR channel is almost certainly not a ClC. Six ClC genes termed clh-1 through -6 are present in the worm genome. Oocytes isolated from worms harboring deletion alleles for clh-1, clh-2, clh-4, or clh-6 expressed normal ICl,OR activity and RT-PCR-confirmed knockdown of clh-5 expression by RNA interference (RNAi) had no effect on ICl,OR amplitude (data not shown). In addition, RNAi directed at clh-3(ok763) mRNA did not alter current properties, indicating that the ICl,OR channel is not encoded by the mutant clh-3 gene (data not shown). Additional functional and molecular studies are required to identify the gene(s) encoding the ICl,OR channel.
As shown in Figs. 8 and 9, ICl,OR activity is dramatically reduced in oocytes undergoing meiotic maturation. Interestingly, CLH-3b and ICl,mec also show striking changes in activity during oocyte and early embryo development. CLH-3b is activated specifically during oocyte meiotic maturation and plays a role in regulating ovulation (19, 26). Membrane expression of CLH-3b is detected throughout all stages of oocyte development (6). However, within minutes after ovulation occurs, the channel is rapidly lost from the plasma membrane and expression of the clh-3 gene is not detected again until very late in embryonic development (unpublished observations). In contrast to CLH-3b, ICl,mec appears to be activated shortly after embryogenesis begins. High levels of ICl,mec activity are present in developing embryo cells as early as the 1- and 2-cell stages, but the current cannot be detected in oocytes (3).
The likely role of CLH-3b is to regulate oocyte membrane potential, which in turn may modulate Ca2+ signaling in the surrounding gap junction coupled sheath cells (19, 26). ICl,OR may also play a role in regulating membrane potential. Such regulation could in turn serve to control oocyte development, ovulation, and/or cell cycle progression. Alternatively, ICl,OR may have a general housekeeping function and participate in intracellular Cl− homeostasis required for proper cell volume and acid-base control. Identification of the gene(s) that encodes the ICl,OR channel will be facilitated by the molecular and genetic tractability of C. elegans and is essential in order to fully define channel function and regulation.
This work was supported by National Institutes of Health (NIH) Grant DK-51610 to K. Strange. Worm strains were obtained from the Caenorhabditis Genetics Center, which is supported by the NIH National Center for Research Resources.
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