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1 Departments of Anaesthesiology, Pharmacology, and Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA2 Department of Medicine, Nephrology Unit, University of Rochester Medical Center, Rochester, NY 14642, USA
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(Received 8 August 2003;
accepted after revision 15 October 2003;
first published online 17 October 2003)
Corresponding author K. Strange: Vanderbilt University Medical Center, T-4202 Medical Center North, Nashville, TN 37232-2520, USA. Email: kevin.strange{at}vanderbilt.edu
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Introduction |
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Six ClC-type anion channel encoding genes termed clh-1–6 or ceclc-1–6 are present in the C. elegans genome (Bargmann, 1998; Schriever et al. 1999; Nehrke et al. 2000). The six nematode ClC channels are representative of the three major subfamilies of mammalian ClCs. We demonstrated recently that C. elegans oocytes functionally express a ClC channel encoded by clh-3 (Rutledge et al. 2001). The biophysical properties and regulation of CLH-3 resemble those of mammalian ClC-2 (reviewed in Strange, 2002; Strange, 2003). CLH-3 is activated during oocyte meiotic cell cycle progression, a process termed meiotic maturation, and in response to oocyte swelling (Rutledge et al. 2001). Activation occurs by serine/threonine dephosphorylation, mediated by the type-1 protein phosphatases CeGLC-7
/ß (Rutledge et al. 2002). Meiotic maturation-induced activation of CLH-3 may function to synchronize oocyte cell cycle progression with ovulation and fertilization (Rutledge et al. 2001; Rutledge et al. 2002; Strange, 2002).
Two CLH-3 splice variants termed CLH-3a and CLH-3b have been cloned from C. elegans (Schriever et al. 1999; Nehrke et al. 2000). These proteins have identical intramembrane domains, but differ significantly in their N- and C-termini. The major differences include a 71 amino acid N-terminal extension on CLH-3a and a 261 amino acid extension of the CLH-3b C-terminus (Fig. 1).
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-helical domains, 17 of which are intramembrane (Dutzler et al. 2002). Dutzler et al. (2002) noted that the C-terminus of the Salmonella enterica ClC participates in Cl- coordination within the channel's selectivity filter. They postulated that this interaction provides a structural basis for regulating channel function via cytoplasmic processes. Consistent with this idea, Fong et al. (1998) showed that replacement of the most C-terminal 290 amino acids of ClC-0 with the corresponding region of ClC-2 prevented slow gating. In addition, replacement of a shorter C-terminal region of ClC-0 with the analogous region in ClC-1 caused a shift in the apparent voltage sensitivity of the channel and induced current attenuation following hyperpolarizing prepulses (Fong et al. 1998). The intracellular N-terminus has also been proposed to function in channel gating. Mutagenesis studies by Jordt & Jentsch (1997) on ClC-2 suggested that the N-terminus plays a role in activation of the channel by cell swelling, voltage and external H+. Although the idea has been challenged recently (Varela et al. 2002), Gründer et al. (1992) proposed that the N-terminus functions as a ‘ball’ that normally blocks the channel pore via a ‘ball-and-chain’ type mechanism, analogous to that of voltage-dependent K+ channels (e.g. Zagotta et al. 1990). A splice variant of ClC-2 with a 10 amino acid deletion in the N-terminus has altered voltage-dependent inactivation kinetics (Cid et al. 2000). Similarly, N-terminal splice variation of ClC-3 alters voltage-dependent inactivation in addition to channel rectification (Shimada et al. 2000).
Given the implied roles of cytoplasmic domains in regulating ClC function, we postulated that the striking differences in the N- and C-termini of CLH-3a and CLH-3b would give rise to channels with distinct gating characteristics and physiological functions. To begin testing this idea, we characterized the biophysical properties of the splice variants expressed heterologously in HEK293 cells. Cell swelling and membrane hyperpolarization activated both channels. However, CLH-3a exhibited unique voltage-dependent characteristics and sensitivity to extracellular Cl- and H+ that was not observed in CLH-3b. The functional properties of heterologously expressed CLH-3b were identical to those of the native Cl- current in the C. elegans oocyte. Immunofluorescence studies using CLH-3b-specific antiserum demonstrated that the channel is expressed in the oocyte plasma membrane. These results indicate that the cell cycle-regulated oocyte Cl- current is probably carried by CLH-3b. Our findings also suggest that the N- and/or C-termini of CLH-3 participate in voltage-dependent gating and influence the channel sensitivity to external Cl- and H+. The differences in functional properties of CLH-3a and CLH-3b suggest that the two channels probably play unique physiological roles in C. elegans.
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Methods |
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Wild type N2 (Bristol) and a clh-3 deletion mutant strain, clh-3(ok763), were cultured at 16°C using standard methods (Brenner, 1974).
Transient expression of CLH-3 splice variants
HEK293 (human embryonic kidney) cells were cultured in Eagle's minimal essential medium (MEM; Gibco, Gaithersburg, MD) containing 10% heat-inactivated fetal bovine serum (Gibco), 50 U ml-1 penicillin and 50 µg ml-1 streptomycin. Cells grown in 35 mm dishes to approximately 50% confluency were transfected using Superfect reagent (Qiagen, Valencia, CA) with either 2 µg green fluorescent protein (GFP) cDNA ligated into pcDNA3, or 1 µg GFP cDNA and 5–6 µg CLH-3a (kindly provided by T. Jentsch) or CLH-3b (Nehrke et al. 2000) cDNA ligated into pCDNA3.1. Cells were transfected for 3 h, washed three times with MEM and incubated overnight at 37°C.
Isolation of C. elegans oocytes
C. elegans gonads were isolated by placing worms in egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM Hepes, pH 7.3, 340 mOsm) and cutting them behind the pharyngeal bulb and in front of the spermatheca using a 26-gauge needle. Isolated gonads were transferred to a patch clamp bath chamber mounted on the stage of an inverted microscope. Late-stage oocytes spontaneously released from the cut end of the gonad were used for patch clamp studies. As described previously (Rutledge et al. 2001), oocytes undergoing meiotic maturation were identified by the presence of an off-centre nucleus and by the disappearance of the nuclear envelope.
Whole-cell patch clamp recording
HEK293 cells were patch clamped 
24 h after transfection. Two hours before initiating electrophysiological experiments, the transfected cells were dissociated by brief exposure to trypsin and then plated onto poly L-lysine-coated cover slips. Plated cover slips were placed in a bath chamber mounted on the stage of an inverted microscope. Cells were visualized by fluorescence and differential interference contrast (DIC) microscopy.
Transfected cells were identified by GFP fluorescence and patch clamped using a bath solution containing 90 mMN-methyl-D-glucamine chloride (NMDG-Cl) or 90 mM NaCl, 5 mM MgSO4, 1 mM CaCl2, 12 mM Hepes, 8 mM Tris, 5 mM glucose, 80 mM sucrose and 2 mM glutamine (pH 7.4, 295 mOsm), and a pipette solution containing 116 mM NMDG-Cl, 2 mM MgSO4, 20 mM Hepes, 6 mM CsOH, 1 mM EGTA, 2 mM ATP, 0.5 mM GTP, and 10 mM sucrose (pH 7.2, 275 mOsm). Low NaCl solutions were prepared by isosmotic substitution of NaCl for sucrose. Cells were swollen by exposure to a hypotonic (225 mOsm) bath solution that contained no added sucrose.
Oocytes were patch clamped using a bath solution containing 116 mM NMDG-Cl, 2 mM CaCl2, 2 mM MgCl2, 25 mM Hepes, and 71 mM sucrose (pH 7.3, 340 mOsm) and a pipette solution containing 116 mM NMDG-Cl, 2 mM MgSO4, 20 mM Hepes, 6 mM CsOH, 1 mM EGTA, 48 mM sucrose, 2 mM ATP, and 0.5 mM GTP (pH 7.2, 315 mOsm). Swelling was induced by exposure to a hypotonic (260 mOsm) bath solution that contained no added sucrose. Low NaCl solutions were prepared by isosmotic substitution of NaCl for sucrose. Oocytes were depleted of ATP by incubation for 20–30 min with 5 mM 2-deoxyglucose and 1 µM rotenone, and patch clamped with an ATP-free pipette solution containing 40 µM oligomycin, 20 µM rotenone and 5 µM iodoacetate. Metabolic inhibitors were dissolved as stock solutions in DMSO and then added to the pipette or bath saline at a final DMSO concentration of 
0.01%.
Patch electrodes were pulled from 1.5 mm outer diameter silanized borosilicate microhaematocrit tubes; electrode resistance ranged from 2 to 4 M
. Currents were measured with an Axopatch 200B (Axon Instruments, Foster City, CA) patch clamp amplifier. Electrical connections to the patch clamp amplifier were made using Ag/AgCl wires and 3 M KCl/agar bridges. Data acquisition and analysis were performed using pClamp 8 software (Axon Instruments).
Measurement of HEK293 cell volume
HEK293 cells were imaged using DIC microscopy during patch clamp experiments. Cell images were recorded using a super VHS video cassette recorder (model SVO-2000; Sony Electronics Inc., San Jose, CA) and a Hamamatsu CCD camera (model C2400; Hamamatsu Photonics K.K., Hamamatsu City, Japan). The cells used for patch clamp experiments had a spherical morphology, and relative cell volume changes were therefore determined as: (experimental CSA/control CSA)3/2, where CSA is the cell cross-sectional area measured at a single focal plane located at the point of maximum cell diameter.
Gonad immunofluorescence
Gravid adult worms were placed in 10 µl of M9 buffer (85.6 mM NaCl, 45.3 mM Na2HPO4, 22 mM KH2PO4, 1 mM MgSO4, pH 7.0, 295 mOsm) on a glass slide, frozen on dry ice and freeze-cracked as described previously (Epstein & Shakes, 1995), then fixed for 10 min each in –30°C methanol and –30°C acetone. Fixed worms were rehydrated for 30 min at room temperature in 0.2% PBS-Tween, blocked with goat serum diluted to 5% with 0.2% PBS-Tween and then exposed to 1: 50–1: 200 dilutions of an affinity purified polyclonal antiserum raised against a fusion protein comprised of the last 132 amino acids (869–1001) of CLH-3b. Following a 60 min incubation, the slides were washed three times in 0.2% PBS-Tween and then treated for 30 min with a 1: 1200 dilution of a Cy3-labelled goat antirabbit secondary antibody. Washed slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and then imaged by DIC and fluorescence microscopy.
RNA interference
Double stranded RNA interference (RNAi) was performed as described previously (Rutledge et al. 2001). Briefly, a DNA template corresponding to the first 847 bp of the open reading frame of clh-3 was obtained by PCR and sense and antisense RNA were synthesized by T7 polymerase (MEGAscript; Ambion, Austin, TX). Template DNA was digested with DNaseI and RNA was precipitated with 3 M sodium acetate and ethanol. Precipitated RNA was washed with 70% ethanol, air-dried and dissolved in water. RNA size, purity and integrity were assayed on agarose gels. dsRNA was formed by annealing sense and antisense RNA at 65°C for 30 min. Annealed dsRNA was diluted into potassium citrate buffer for injection. Worms were injected into one gonad arm with approximately 1000 000 molecules of dsRNA.
Statistical analyses
Data are presented as means ±S.E. Statistical significance was determined using Student's two-tailed t test for paired or unpaired means. P-values of 0.05 were taken to indicate statistical significance.
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Results |
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Figure 1 shows the amino acid sequence alignment of CLH-3a and CLH-3b. Identical regions of the two splice variants are shaded grey and non-identical regions are shaded yellow. Sequence outlined in black and red denote cystathionine-ß-synthase (CBS) domains and presumed intracellular N- and C-termini respectively. Note that CLH-3a and CLH-3b are identical with the exception of their N- and C-terminal cytoplasmic domains.
The functional characteristics of CLH-3 splice variants heterologously expressed in HEK293 cells were evaluated using whole-cell patch clamp recording techniques. Figure 2A shows current traces recorded from GFP-positive cells cotransfected with GFP and channel cDNA. Both CLH-3a and CLH-3b were constitutively active and produced robust currents that exhibited strong inward rectification and time-dependent activation at hyperpolarized test potentials. Untransfected cells (n= 8) or cells transfected with GFP alone (n= 4) exhibited no hyperpolarization-activated currents (data not shown).
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We were unable to assess the voltage dependence of channel activation for the two splice variants using tail current analysis. As reported previously for CLH-3a expressed in Xenopus oocytes (Schriever et al. 1999), we observed no tail currents for either variant due to rapid channel inactivation at positive potentials. We therefore attempted to use a negative test pulse for tail current analysis. However, the maximum apparent channel open probability of both splice variants was more negative than –170 mV (data not shown), and voltage clamping HEK293 cells beyond this voltage was difficult due to the instability of the cell membrane. Furthermore, it was not possible to estimate the minimal channel open probability for CLH-3a due to potentiation by conditioning voltages more positive than –40 mV (see below). We therefore assessed the activation characteristics of CLH-3a and CLH-3b from normalized current values fitted with a Boltzmann relation (Fig. 2C). The estimated half-activation potential (V0.5) values derived from the Boltzmann fits for CLH-3a and CLH-3b were –99 ± 2 mV (n= 11) and –87 ± 3 mV (n= 11) respectively. The slope factors, k, for CLH-3a and CLH-3b were 10 ± 2 mV-1 and 18 ± 3 mV-1 respectively.
The activation kinetics of CLH-3a were considerably slower than those of CLH-3b. Figure 3A shows current traces recorded at –120 mV for the two channels. The CLH-3a current (ICLH-3a) trace has been scaled by a factor of 1.7 and superimposed over the CLH-3b current (ICLH-3b) trace. The time constants for activation were determined by fitting the current traces with exponential functions. ICLH-3a and ICLH-3b were well fitted by double exponentials during the first 50 ms of hyperpolarization-induced channel activation. As shown in Fig. 3B, the slow time constant, 
1, was similar for both channels and voltage independent (P > 0.5). The fast time constant, 2, for CLH-3a was two- to threefold greater (P < 0.01) than that of CLH-3b and was voltage independent between –120 mV and –80 mV (P > 0.17; Fig. 3B). Over this same voltage range, 2 for CLH-3b showed a small but significant (P < 0.01) increase with depolarization (Fig. 3B).
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Schriever et al. (1999) reported that hyperpolarization-induced activation of CLH-3a expressed in Xenopus oocytes is potentiated by conditioning predepolarization. We therefore tested whether a similar voltage clamp protocol altered CLH-3a and CLH-3b activation in HEK293 cells. Transfected cells were held at 0 mV and then voltage clamped for 1–7 s at 60 mV before stepping to –120 mV for 2 s to activate the channels. As shown in Fig. 4A, ICLH-3a activity was potentiated by predepolarization of HEK293 cells. The extent of potentiation increased as a function of the time the cell was clamped at 60 mV (Fig. 4A and B). In contrast, ICLH-3b was not potentiated by predepolarization. Instead, hyperpolarization-induced channel activity actually decreased slightly by 5% when cells were clamped at 60 mV for 7 s (Fig. 4A and B).
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We also found that conditioning depolarization induced inactivation of ICLH-3a (Fig. 5A). Figure 5C summarizes the voltage dependence of inactivation for the two splice variants. The data are presented as fractional current, where the mean pseudo steady-state current amplitude (ISS) measured during the last 20 ms of the –120 mV test pulse is divided by the peak current amplitude (Ipeak). ICLH-3a inactivation was rarely observed following a conditioning potential of –20 mV, but became evident with stronger depolarizing prepotentials (Fig. 5C). For example, predepolarization to 60 mV caused ICLH-3a to inactivate by 21 ± 2% over the 2 s, –120 mV test potential. In contrast, conditioning predepolarization failed to induce ICLH-3b inactivation (Fig. 5A and C).
The time constants that describe ICLH-3a activation and inactivation as a function of conditioning potential are shown in Fig. 6. Activation time constants were derived by fitting a double exponential to the first 50 ms of hyperpolarization-induced channel activation. The inactivation process was well described by fitting a single exponential to the entire time course of inactivation. The conditioning potential had no significant (P > 0.12) effect on any of the time constants.
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Voltage-dependent gating of some ClC channels is coupled to extracellular Cl-, and reductions in Cl- concentration decrease channel open probability (reviewed in Jentsch et al. 2002). We therefore characterized the effect of external Cl- concentration on ICLH-3a and ICLH-3b. Whole-cell currents were recorded under control conditions (92 mM Cl-) and at 30 s after switching to a low Cl- (12 mM) bath solution. In the absence of regulatory effects, reductions in extracellular Cl- concentration are expected to increase inward current due to an increase in the outward driving force on the ion. Figure 7A and B show normalized I–V relationships for CLH-3a and CLH-3b recorded in control and low Cl- bath solutions. Reduction of the bath Cl- significantly (P < 0.01) inhibited ICLH-3a at test potentials of –80 mV to –120 mV (Fig. 7A), but had no effect (P > 0.3) on ICLH-3b (Fig. 7B). As summarized in Fig. 7C, mean ICLH-3a amplitude at –100 mV was 1.8-fold greater in the presence of 92 mM Cl-versus 12 mM Cl-. This effect was due specifically to reduction of the bath Cl- as replacement of NaCl with NMDG-Cl had no significant (P = 0.98) effect on ICLH-3a amplitude (n= 3; data not shown).
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13-fold as extracellular H+ concentration was increased from pH 8.1 to pH 5.9. In contrast, CLH-3b activity increased by only 2.2-fold over this pH range (Fig. 8C).
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Regulation of CLH-3 splice variants by cell swelling
Hypotonicity-induced cell swelling activates native CLH-3 expressed in C. elegans oocytes (Fig. 9 and Rutledge et al. 2001). To determine whether CLH-3 splice variants exhibit differences in their volume sensitivity, we measured CLH-3a and CLH-3b current amplitude in transfected HEK293 cells 1 min after reducing bath osmolality from 295 mOsm to 225 mOsm. Cell swelling during this time period is insufficient to activate the ubiquitous outwardly rectifying Cl- current ICl,swell (Rutledge et al. 2002).
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20%. Mean ±S.E. relative whole-cell currents in hypotonically swollen CLH-3a- and CLH-3b-transfected cells were 1.19 ± 0.04 (n= 4) and 1.23 ± 0.06 (n= 4) respectively. These values were not significantly (P > 0.5) different. Biophysical characteristics of the native oocyte Cl- current
The existence of two CLH-3 splice variants with unique structural and biophysical properties suggests that the channels perform distinct physiological roles. To begin addressing this important issue, we carried out a detailed biophysical characterization of the native oocyte Cl- current.
Figure 9A shows whole-cell currents in a non-maturing wild-type oocyte under basal conditions and after maximal current activation induced by hypotonic swelling for 9 min. Also shown are whole-cell currents in an oocyte isolated from a clh-3(ok763) mutant worm. The clh-3(ok763) oocyte was swollen for 10 min. I–V relationships for the three whole-cell currents are shown in Fig. 9B. No inwardly rectifying Cl- current was observed in clh-3(ok763) oocytes under basal conditions or after prolonged (8–15 min) swelling (n= 7). The ok763 allele is an 1500 bp deletion in the clh-3 gene. The absence of hyperpolarization-induced Cl- currents in clh-3(ok763) oocytes demonstrates that both basal and swelling-activated inwardly rectifying Cl- channels are encoded by clh-3, a finding that is consistent with our previous RNAi studies (Rutledge et al. 2001).
We measured the time constants of activation of nativeICLH-3 at various test voltages. To facilitate comparison of the native current with heterologously expressed CLH-3a and CLH-3b, we measured slow (1) and fast (2) time constants by fitting a double exponential to the first 50 ms of hyperpolarization-induced current activation. Figure 9C shows the time constants plotted as a function voltage. Both the 1 and 2 of native ICLH-3 were similar to those of ICLH-3b and showed similar voltage dependencies. The constant 1 was voltage insensitive (P > 0.5) and 2 showed a small but significant (P < 0.002) increase with depolarization (n= 9).
Figure 10A shows the voltage dependence of predepolarization on native ICLH-3 when activated by oocyte swelling. The oocyte was voltage clamped for 3 s at conditioning potentials of –20 mV to 60 mV and then stepped to -120 mV for 2 s. No potentiation of the current was observed at any of the voltages tested.
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To assess whether prolonged depolarization would potentiate native ICLH-3, we measured the effect of voltage clamping swollen oocytes to 60 mV for 1–7 s. As shown in Fig. 10C and D, native ICLH-3 was not potentiated by depolarization. Instead, current amplitude declined slightly as a function of the time oocytes were depolarized. Following a 7 s predepolarization to 60 mV, native ICLH-3 amplitude was reduced by 7% (Fig. 10D). This small decrease in current is similar to that observed for ICLH-3b in HEK293 cells (Fig. 4B).
We also characterized the effects of extracellular Cl- and pH on native ICLH-3. Whole-cell current was measured in the presence of control saline containing 124 mM Cl- and at 30 s after switching to a 16 mM Cl- bath solution. This represents an 87% reduction in extracellular Cl-, which is identical to the change in bath Cl- used to evaluate the heterologously expressed CLH-3 splice variants. Figure 11A shows the mean ±S.E.I–V relationships for native ICLH-3 activated by swelling in 124 mM and 16 mM Cl- bath solutions. Reduction of the bath Cl- had no significant (P > 0.1) effect on native ICLH-3 at test potentials of –100 mV to –60 mV. However, at -40 mV, reduction of the bath Cl- caused a small but significant (P < 0.01) increase in native ICLH-3 amplitude. The lack of change in current amplitude at –100 mV during reduction of the bath Cl- is identical to that observed for ICLH-3b (Fig. 7C).
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2-fold, similar to that observed for ICLH-3b (Fig. 8C). As shown in Fig. 11C, the voltage dependence of the acidification effect was weak and similar to that of ICLH-3b (Fig. 8D). At -40 mV, the mean ±S.E. acidification-induced increase in current amplitude was 1.22 ± 0.05-fold (n= 7) greater than that observed at –100 mV. The mean ±S.E. relative acidification-induced increase of ICLH-3b at -40 mV versus-100 mV was 1.52 ± 0.09-fold (n= 8).
Immunolocalization of CLH-3b in C. elegans oocytes
The C. elegans adult hermaphrodite gonad consists of two U-shaped arms connected to a common uterus. The distal gonad contains germline nuclei embedded in a syncitial cytoplasm. In the loop region of the gonad, a plasma membrane forms around these nuclei in a process termed ‘cellularization’. Newly formed oocytes develop in the proximal region of the gonad and accumulate in a single row of graded developmental stages. The most differentiated oocyte is positioned adjacent to the spermatheca where it undergoes meiotic maturation and is then ovulated into the spermatheca and fertilized (reviewed by Hubbard & Greenstein, 2000).
The native oocyte Cl- current has biophysical properties virtually indistinguishable from those of heterologously expressed CLH-3b. To determine whether CLH-3b is translationally expressed in oocytes, we generated a polyclonal antiserum against the last 132 amino acids of the channel protein. Isolated gonads were fixed and immunostained using this antiserum and a Cy3-labelled secondary antibody. As shown in Fig. 12A and B, CLH-3b was detected in the plasma membrane at all stages of oocyte development, including newly formed oocytes in the loop region of the gonad. A faint cytoplasmic and occasionally a nuclear background staining were observed when gonads were reacted with the preimmune serum or with antiserum that had been previously competed with an 85-fold molar excess of the CLH-3b fusion protein (data not shown). Gonads in which CLH-3 expression was disrupted by RNAi showed faint cytoplasmic and nuclear staining (Fig. 12C) similar to that observed with the preimmune serum or the CLH-3b fusion protein-competed antiserum. These results demonstrate that CLH-3b is expressed in the C. elegans oocyte beginning at very early stages of oocyte development when the plasma membrane is first formed around germline nuclei.
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Discussion |
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Alternative splicing increases the functional diversity of numerous channel types (Fettiplace & Fuchs, 1999; Lipscombe et al. 2002). However, almost nothing is known about the role of alternative splicing in ClC function and regulation. We have demonstrated in this work that splice variation of the predicted intracellular N- and C-termini of CLH-3 causes significant changes in channel biophysical properties.
Both CLH-3a and CLH-3b are activated by cell swelling and membrane hyperpolarization. However, the splice variants exhibit significant differences in activation kinetics and voltage sensitivity. Hyperpolarization-induced activation of CLH-3b is approximately threefold faster than that of CLH-3a (Fig. 3A and B). In addition, CLH-3b is active at physiologically relevant membrane voltages (i.e. -40 mV to -60 mV) whereas CLH-3a requires much stronger hyperpolarization for activation (Fig. 2B and C).
A particularly striking difference between CLH-3a and CLH-3b is their sensitivity to conditioning voltages. As reported previously (Schriever et al. 1999), we found that predepolarization activated CLH-3a and induced slow inactivation of the current at hyperpolarized potentials (Figs 4 and 5). Schriever et al. (1999) showed that increased extracellular Cl- activates CLH-3a during the depolarizing prepulse and that the degree of predepolarization-induced activation is dependent on the concentration of the bath Cl-. They further demonstrated that activation of CLH-3a by depolarizing prepulses and increased extracellular Cl- had similar time courses. The effect of predepolarization on CLH-3a was attributed to a slow gating process that is regulated by both membrane potential and extracellular Cl- (Schriever et al. 1999).
It is conceivable that membrane potential per se does not regulate the putative CLH-3a slow gate. Instead, prolonged depolarization may raise local Cl- concentration near the proposed Cl- binding site that is thought to regulate ClC gating (Chen & Miller, 1996; Dutzler et al. 2003). In this regard, it is interesting to note that predepolarization has almost no effect on the amplitude of ICLH-3b (Figs 4 and 5) and the channel is relatively insensitive to changes in the bath Cl- concentration (Fig. 7B-C). The insensitivity of CLH-3b to depolarizing prepulses may be due to a relative insensitivity of the gating mechanism to extracellular Cl-.
We found that of the two splice variants, CLH-3a is activated to a much greater degree by extracellular acidification (Fig. 8). The effect of extracellular pH on ClC channel activity has been attributed either to a direct effect of extracellular H+ on the activation gate (Rychkov et al. 1996; Rychkov et al. 1997; Dutzler et al. 2003), or to changes in surface charge resulting from H+ binding to anionic sites in or near the channel pore (Arreola et al. 2002; Ferroni et al. 2000; Jordt & Jentsch, 1997). One possible explanation for the difference in pH sensitivity of CLH-3a and CLH-3b is that splice variation may alter the H+ affinities and/or accessibility of titratable residues that regulate channel activation. While we cannot define at present the mechanism by which acidification activates CLH-3, the differences in the effects of predepolarization, extracellular Cl- concentration and pH on the splice variants suggest that all three may act on a single gating mechanism that differs between the two channels.
How might alternative splicing of presumed intracellular N- and C-termini affect the sensitivity of CLH-3 to predepolarization and extracellular ions?Dutzler et al. (2002,2003) recently identified in the high-resolution crystal structures of bacterial ClC channels, a highly conserved glutamate residue in the outer channel pore that may serve as a Cl--and H+-dependent activation gate. In the closed state, the glutamate carboxyl group is thought to occlude the channel pore by projecting into the selectivity filter. During channel activation, the carboxyl group would be displaced out of the filter to allow ion conduction. Dutzler et al. (2003) suggested that extracellular Cl- and H+ activate bacterial ClC channels by displacing the residue. To support their model, Dutzler et al. (2003) demonstrated that mutation of the corresponding glutamate residue in ClC-0 causes constitutive channel activation.
Interestingly, Dutzler et al. (2002) showed that the -helix that immediately precedes the intracellular C-terminus of bacterial ClC channels contributes directly to the coordination of Cl- within the selectivity filter. They speculated that the C-terminus could provide a direct route for regulating channel activity by intracellular signalling events. Alignment of CLH-3 with the Salmonella typhimurium ClC channel revealed that the glutamate residue identified in the X-ray crystal structures is conserved in both splice variants. In addition, isoleucine, phenylalanine, serine and tyrosine residues, which are thought to function in Cl- binding within the bacterial ClC channel pore (Dutzler et al. 2002), are also conserved. If these conserved residues are involved in ion conduction and activation of CLH-3, the different sensitivities of CLH-3a and CLH-3b to predepolarization, bath Cl- and pH suggests that alternative splicing of the C-terminus may affect the accessibility and/or function of pore-associated ion binding sites. Recent electrophysiological studies carried out on oocytes that were isolated from a worm strain harbouring a deletion mutation in the CLH-3 C-terminus strongly support this idea (J. Denton & K. Strange, unpublished results). Detailed molecular analysis of CLH-3a and CLH-3b is clearly warranted to explore how C-terminal splice variation alters channel gating.
In addition to a role in regulating channel gating, it is intriguing to speculate that the unique C-terminus of CLH-3b may also play an important role in cell cycle signalling mechanisms. We have recently identified four proteins by yeast 2-hybrid analysis that interact with this region of the channel (J. Denton, K. Nehrke & K. Strange, unpublished results). Two of the four proteins may play well-established roles in cell cycle functions. A third protein is a component of a putative protein complex identified by large-scale protein–protein interaction mapping (Walhout et al. 2000). This protein complex includes two MAP kinases known to play essential roles in C. elegans meiotic cell cycle progression. Thus, the C-terminus of CLH-3b may function as a scaffold for proteins that regulate channel activity and/or other cell cycle-associated physiological processes.
Possible physiological roles of CLH-3 splice variants
The striking differences in their structure and biophysical properties suggest strongly that CLH-3 splice variants perform specific physiological roles in different cell types. GFP reporter studies have demonstrated that clh-3 is transcriptionally expressed in the excretory cell, vulval cells, uterus, hermaphrodite-specific neurones (HSN), enteric muscles, and the first four epithelial cells of the intestine (Schriever et al. 1999; Nehrke et al. 2000). Schriever et al. (1999) suggested that depolarization-induced potentiation of CLH-3a (Figs 4 and 5) may play an important role in excitable cell function. For example, enteric muscles contract during the expulsion phase of the defecation cycle (Thomas, 1990; Liu & Thomas, 1994) and appear to be excited directly by the GABAergic motor neurones AVL and DVB (McIntire et al. 1993). These muscle cells express L-type Ca2+ channels that likely give rise to action potentials with relatively long-lived periods of depolarization (Lee et al. 1997). If CLH-3a is expressed in the enteric muscles, hyperpolarization-induced activation would be potentiated by prolonged depolarization (Figs 4 and 5). During repolarization, CLH-3a opening could conceivably function to correct overshoots in membrane voltage (see, for example, Davis et al. 1999).
In contrast to muscle cells, relatively little is known about the electrophysiological properties of specific types of C. elegans neurones. Goodman et al. (1998) developed a so-called ‘slit worm preparation’ where neurones are exposed by cutting a small slit in the animal's cuticle. These studies suggested that nematode neurones do not exhibit classical rapid and repetitive action potentials and that instead they transmit electrical signals via a passive spread of membrane voltage. As in muscle cells, CLH-3a could function in neurones to correct overshoots in membrane voltage during repolarization. Interestingly, CLH-3 expression appears to be limited largely to the HSN, which innervate vulval muscles and control egg laying (Trent et al. 1983). No electrophysiological measurements have been carried out on the HSN, but it is conceivable that their electrophysiological properties differ from the neurones studied by Goodman et al. (1998) and may depend uniquely on CLH-3 activity.
As shown in the present study, CLH-3b is expressed in the oocyte (Fig. 12) and is probably responsible for the inwardly rectifying whole-cell current (e.g. Fig. 9). The physiological stimulus for activation of CLH-3b is initiation of oocyte meiotic maturation (Rutledge et al. 2001; Rutledge et al. 2002). Signals released from the maturing oocyte induce contractions in the surrounding smooth muscle-like sheath cells that drive the oocyte into the spermatheca (McCarter et al. 1999). Knockdown of clh-3 expression by RNAi disrupts the timing of sheath cell ovulatory contractions (Rutledge et al. 2001). We have suggested that activation of CLH-3 depolarizes the oocyte and electrically coupled sheath cells and that depolarization in turn modulates Ca2+ signalling pathways that control sheath contractility (Rutledge et al. 2001; Strange, 2002; Strange, 2003).
Depolarization-induced potentiation such as is observed with CLH-3a (Figs 4 and 5) would probably have no functional significance in the oocyte. Thus, expression of the CLH-3b splice variant may be restricted to oocytes and other nonexcitable cells such as the intestine and excretory cell. The primary function of CLH-3b in the oocyte may be to depolarize membrane potential. In the intestine and excretory cell, CLH-3b may function in transepithelial Cl- transport. Immunolocalization of CLH-3a and CLH-3b expression patterns coupled with whole animal functional assays and electrophysiological studies in cultured C. elegans cells (Christensen et al. 2002) should help to elucidate the precise physiological roles of these splice variants.
In conclusion, we have shown that alternative splicing of the CLH-3 N- and C-termini has striking effects on the biophysical properties of this channel. Identification of the structural basis of these effects should provide new understanding of the molecular mechanisms of ClC gating and the role of intracellular domains in regulating ClC channel activity. Furthermore, the detailed characterization of CLH-3a and CLH-3b expression patterns and the physiology of CLH-3-expressing cells will likely yield novel insights into anion channel function and the role of splice variation in increasing ClC functional diversity.
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