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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Departments of Anesthesiology, Molecular Physiology and Biophysics, and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee; and 2Nephrology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, New York
Submitted 22 August 2005 ; accepted in final form 18 November 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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C, in its native cellular environment. CLH-3bC exhibits altered voltage-dependent gating as well as pH and Cl– sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn2+, prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs. chloride; cell volume; voltage-gated anion channel
-helical domains, 17 of which are intramembrane (12). Monomers form a single, independently gated pore or protopore. Protopore gating is a fast process and is thought to be mediated by the movement of a highly conserved glutamate residue located adjacent to the selectivity filter (13) and perhaps larger-scale structural rearrangements of the pore (1). Many ClC channels also exhibit a slow or common gating process that opens and closes the two protopores simultaneously (Refs. 17, 22; for review, see Ref. 20). The molecular basis of slow gating is unknown but may involve large-scale channel conformational changes (5, 26, 31).
Eukaryotic ClCs have extensive cytoplasmic NH2 and COOH termini that are absent from crystallized bacterial ClC proteins. In addition, the COOH termini of all eukaryotic ClC channels contain two cystathionine-
-synthase (CBS) domains (3, 20). The bacterial ClC crystal structures indicate that the last -helix immediately preceding the cytoplasmic COOH terminus contributes directly to the coordination of Cl– within the channel selectivity filter (12). Dutzler et al. (12) postulated that the interaction of this -helix with cytoplasmic structures could provide a mechanism for regulating channel-gating properties and activity. Consistent with this idea, mutagenesis studies have demonstrated that cytoplasmic NH2 and COOH termini play important roles in ClC gating (8, 15, 16, 19, 30). COOH-terminal mutations in ClC-1 and ClC-2 are associated with myotonia and epilepsy (4, 18, 25). Recent studies have suggested that CBS domains may function in slow gating (14). However, despite their obvious importance, the precise structural and functional relationships of cytoplasmic NH2 and COOH termini are poorly defined.
Six ClC-type anion channel-encoding genes, termed clh-1–clh-6 or ceclc-1–ceclc-6, are present in the Caenorhabditis elegans genome (2, 23, 29). The six nematode ClC genes are representative of the three major subfamilies of mammalian ClC genes. Two CLH-3 splice variants, termed CLH-3a and CLH-3b, have been cloned from C. elegans (23, 29). These proteins have identical intramembrane domains but differ significantly in their cytoplasmic NH2 and COOH termini. The major differences include a 71-amino acid NH2-terminal extension on CLH-3a and a 270-amino acid extension of the CLH-3b COOH terminus. CLH-3b is expressed in the C. elegans oocyte and gives rise to a swelling- and meiotic cell cycle-regulated Cl– current (9).
CLH-3a and CLH-3b exhibit striking differences in voltage sensitivity, activation kinetics, and sensitivity to extracellular pH and Cl– concentration (9). On the basis of structural and functional insights gained from crystallized bacterial ClC proteins (12, 13), we postulated that alternative splicing of the COOH terminus may account for these differences by altering the accessibility and/or function of pore-associated ion-binding sites (9).
We recently identified a worm strain harboring an 841-nt deletion in a region of clh-3 encoding the predicted cytoplasmic COOH termini of CLH-3a and CLH-3b. The deletion removes 101 amino acids that are unique to CLH-3b and another 64 amino acids shared by both splice variants. Mutant CLH-3b channels are functionally expressed in the C. elegans oocyte. To begin defining the role of cytoplasmic domains in regulating CLH-3b gating and activity, we characterized the biophysical properties of the mutant channels in their native cellular environment. We have demonstrated in the present study that the deletion mutation of the COOH terminus disrupts volume- and phosphorylation-dependent channel regulation and dramatically alters voltage sensitivity, sensitivity to extracellular H+ and Cl–, and inhibition by Zn2+. These studies provide new insights into the role of the cytoplasmic domains in the regulation of ClC gating and activity and provide the foundation for future site-directed mutagenesis and heterologous expression studies.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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PCR analysis. clh-3(ok768) mutant worms were outcrossed three times, and homozygous animals were identified using PCR analysis of genomic DNA isolated from single worms (24) and the primer set 5'-GTCAATTCGCTCATTCGGTT-3' and 5'-GGTCAAGAAACGGAAAACCAA-3' (Fig. 1A). To determine the mutant mRNA sequence, oligo(dT) primed cDNA was prepared from RNA isolated from Bristol N2 and clh-3(ok768) worms using standard techniques. A partial open reading frame was PCR amplified, cloned, and sequenced using the primer set 5'-CCTGATATTCCCCATACAACGTCC-3' and 5'-TCAGAATTTTTCGTCATGAAC-3'. These primers annealed in exon 11 and exon 19 immediately before the stop codon (see Fig. 1B).
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Patch-clamp electrodes were pulled from 1.5-mm-outer-diameter silanized borosilicate microhematocrit tubes. Currents were measured using an Axopatch 200B (Axon Instruments, Foster City, CA) patch-clamp amplifier with control bath solution containing (in mM) 116 NMDG-Cl, 2 CaCl2, 2 MgCl2, 25 HEPES, and 71 sucrose (pH 7.3; 340 mosM) and a pipette solution containing (in mM) 116 NMDG-Cl, 2 MgSO4, 20 HEPES, 6 CsOH, 1 EGTA, 48 sucrose, 2 ATP, and 0.5 GTP (pH 7.2; 315 mosM). For low-Cl– experiments, we used a NaCl-based bath solution that was otherwise identical to the NMDG-Cl– bath solution. Low-NaCl solutions were prepared by isosmotic substitution of sucrose for NaCl. Oocytes were swollen by exposure to a hypotonic (260 mosM) bath solution that contained no added sucrose. Metabolic inhibitors were dissolved as stock solution in DMSO and then added to pipette or bath solutions at a final DMSO concentration of 
0.01%.
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).
RNA interference.
RNA interference (RNAi) was performed as described previously (27). 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 using T7 polymerase (MEGAscript; Ambion, Austin, TX). Template DNA was digested with DNase I, and RNA was precipitated using 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. Double-stranded RNA (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 in one gonad arm with 
1,000,000 molecules of dsRNA.
Statistical analysis.
Data are presented as means ± SE. Statistical significance was determined using Student's two-tailed t-test. P 0.05 was assumed to indicate statistical significance.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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800-bp deletion (Fig. 1A). DNA sequencing demonstrated that the deletion spanned a region of 841 nt that included part of exon 12 and all of exons 13 and 14, which are present only in CLH-3b (23) (Fig. 1B). PCR analysis of the mutant mRNA demonstrated that it coded for a protein where a cryptic site in exon 12 was spliced in frame to exon 15 (Fig. 1B). Amplification using primers specific for CLH-3a did not yield a PCR product (data not shown).
The mutant transcript lacked sequence coding for COOH-terminal amino acids V604–E768 of the CLH-3b protein (Fig. 1C). We termed the deletion mutant protein CLH-3bC. Amino acids R668–E768 are unique to CLH-3b and are encoded by exons 13 and 14. Amino acids V604–S667 comprise a domain that is conserved in both CLH-3a and CLH-3b (Fig. 1C). Eleven of the amino acids deleted in this region are present in the first CBS domain.
CLH-3bC forms channels with altered voltage sensitivity and activation kinetics.
To determine whether CLH-3bC channels are functional, we recorded whole cell Cl– currents in oocytes isolated from wild-type and clh-3(ok768) worms. As shown previously (9, 27, 28), wild-type meiotic cell cycle-arrested, nonswollen oocytes exhibit a small inwardly rectifying CLH-3b current (ICLH-3b) that is activated by strong hyperpolarization. Figure 2A shows mean whole cell current traces recorded in nine wild-type oocytes. The mean amplitude of this current recorded during the last 20 ms of the 1-s test pulse to –100 mV was –13 pA/pF (Fig. 2B).
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C current (ICLH-3bC) amplitude measured during the last 20 ms of the –100-mV test pulse was –26 pA/pF (Fig. 2B), which was significantly (P < 0.02) greater than that observed in wild-type worms.
To test whether this current was due to the activity of a clh-3-encoded channel, we performed RNAi experiments. clh-3 RNAi disrupts CLH-3 channel expression in the C. elegans oocyte (9, 27, 28). Steady-state current amplitude recorded at –100 mV in oocytes isolated from clh-3(ok768) worms injected with clh-3 dsRNA was inhibited >95% (P < 0.01) to a mean ± SE value of –1.2 ± 0.8 pA/pF (n = 3). These results demonstrate that whole oocyte Cl– currents in clh-3(ok768) worms are carried by CLH-3bC channels.
The kinetics of hyperpolarization-induced activation of ICLH-3bC were considerably more rapid than those of ICLH-3b. Figure 2C shows average current traces recorded at –100 mV from wild-type and clh-3(ok768) oocytes. The ICLH-3b trace is scaled by a factor of 2.1 and superimposed over the ICLH-3bC trace to facilitate comparison of their activation kinetics. In nonswollen, cell cycle-arrested oocytes, ICLH-3b activation can be described by a monoexponential function. The mean ± SE time constant for ICLH-3b activation at –100 mV was 942 ± 190 ms (n = 9). By contrast, ICLH-3bC activation was a biexponential process described by fast and slow time constants at –100 mV of 13 ms and 58 ms, respectively (discussed in detail below).
As shown previously (27, 28), oocyte swelling dramatically increases ICLH-3b amplitude. Figure 3A shows that hypotonicity induced oocyte swelling for 10-min-activated ICLH-3b 23-fold. In marked contrast, ICLH-3bC amplitude increased only 1.2-fold in response to oocyte swelling for 10 min (Fig. 3A).
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C amplitude.
Oocyte shrinkage inhibits ICLH-3b that has been activated by either oocyte swelling or meiotic cell cycle progression (27). However, ICLH-3bC amplitude was not significantly (P > 0.8) altered by cell shrinkage. Mean ± SE relative current amplitude measured at –100 mV after 10-min exposure of clh-3(ok768) oocytes to hypertonic saline was 0.98 ± 0.08 (n = 6). We conclude that the COOH-terminal deletion mutation drastically reduces CLH-3b volume sensitivity, prevents dephosphorylation-induced channel activation, and alters the kinetic properties of the channel.
Oocyte swelling alters the voltage sensitivity and kinetics of hyperpolarization-induced activation of ICLH-3b (9, 27). Figure 4A shows mean whole cell currents recorded in wild-type and clh-3(ok768) oocytes swollen for 10 min. Mean CLH-3b and CLH-3bC current densities at –100 mV were –175 pA/pF and –25 pA/pF, respectively.
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C recorded in swollen oocytes were considerably different. Figure 4B shows Boltzmann relationships for ICLH-3b and ICLH-3bC. As described previously by us (9) and by Schriever et al. (29), CLH-3 channels close too rapidly at depolarized potentials for tail currents to be measured reliably. Furthermore, the depolarization-induced potentiation of ICLH-3bC (see below) prevented tail current analysis using a negative test pulse. Consequently, we were unable to estimate the voltage dependence of CLH-3bC open probability using tail current analysis. We therefore normalized steady-state current values recorded at each test potential to those measured at –100 mV and fitted this relationship using a Boltzmann function. As shown in Fig. 4B, ICLH-3b was activated at more depolarized potentials than ICLH-3bC, causing a rightward shift in its current-voltage (I-V) relationship. The mean ± SE half-activation voltages (V0.5) and slope factors (k) derived from the Boltzmann curves for ICLH-3b and ICLH-3bC were –67 ± 0.7 mV and 17 ± 0.6 mV–1 (n = 9) and –80 ± 0.4 mV (n = 6) and 9 ± 0.4 mV–1 (n = 6), respectively. Similar V0.5 and k values for ICLH-3bC were observed in nonswollen oocytes (data not shown).
The kinetics of channel activation were estimated by fitting two-term exponential functions that described 
fast and slow components of the activation portion of current traces. Because of the small current amplitude of ICLH-3bC at –60 mV, reasonable exponential fits could be performed only at –80 mV and –100 mV. The fast values for both currents were voltage dependent and increased significantly (P < 0.0007) with depolarization (Fig. 4C). In contrast, slow values for ICLH-3b and ICLH-3bC were voltage independent (P > 0.2) over the voltage range tested (Fig. 4D).
We also observed statistically significant (P < 0.0001) differences in fast and slow values of ICLH-3b compared with ICLH-3bC. However, because the amplitude of ICLH-3b is much greater than that of ICLH-3bC (Fig. 4A), the relevance of such apparent differences is unclear at present.
CLH-3bC exhibits predepolarization-induced potentiation.
The slower activation kinetics and hyperpolarizing shift in the I-V relationship of ICLH-3bC are reminiscent of the biophysical characteristics of heterologously expressed CLH-3a (9). A striking characteristic of CLH-3a is its sensitivity to conditioning predepolarization. Both we (9) and Schriever et al. (29) showed previously that hyperpolarization-induced activation of CLH-3a is potentiated by prior membrane depolarization. However, native CLH-3b in the nematode oocyte as well as heterologously expressed CLH-3b are virtually insensitive to depolarizing prepulses (9). Given the similarities of voltage-dependent activation of CLH-3bC and CLH-3a, we tested the effect of conditioning predepolarization on ICLH-3bC. Oocytes isolated from clh-3(ok768) worms were clamped for 3 s at conditioning potentials between –20 mV and +60 mV in 20-mV increments and then stepped to –100 mV for 2 s to activate ICLH-3bC. Figure 5A shows that hyperpolarization-induced activation of ICLH-3bC was potentiated by conditioning predepolarization. For example, ICLH-3bC amplitude recorded at –100 mV after a conditioning potential of 60 mV was 1.5-fold that after a conditioning potential of –20 mV (Fig. 5B).
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C. Figure 5C summarizes the voltage dependence of inactivation of ICLH-3bC. The degree of inactivation was calculated by normalizing the pseudo-steady-state current (Iss) amplitude recorded during the last 20 ms of the –100-mV test pulse to the peak current (Ipeak) amplitude recorded between 170 and 270 ms. This time domain was chosen because it bracketed the peak ICLH-3bC amplitude after depolarized conditioning pulses (see Fig. 5A). ICLH-3bC inactivation was rarely observed after a conditioning potential of –20 or 0 mV but became prominent with stronger depolarizing prepulses (Fig. 5C). After a conditioning pulse to +60 mV, ICLH-3bC was inactivated by 15% after the current was activated by hyperpolarization to –100 mV.
CLH-3bC exhibits altered sensitivity to extracellular Cl– and pH.
Heterologously expressed CLH-3a is significantly more sensitive than CLH-3b to changes in extracellular Cl– and H+ concentration (9). We therefore examined the effects of changes in bath Cl– concentration and pH on ICLH-3bC. Whole cell currents in clh-3(ok768) oocytes were recorded in control bath (124 mM Cl–) and 30 s after switching to a 16 mM Cl– bath solution. Figure 6A shows mean ± SE currents recorded between –100 mV and +40 mV in control and low-Cl– bath solutions. Reduction of Cl– inhibited ICLH-3bC. At –100 mV, for example, ICLH-3bC amplitude was reduced 24% (P < 0.0001). A similar change in bath Cl– concentration has no effect on heterologously expressed ICLH-3b or ICLH-3b measured in wild-type oocytes (9).
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C. Reduction of bath pH from 8.1 to 5.9 increased steady-state ICLH-3bC amplitude 6.3-fold at –100 mV. In contrast, a similar reduction of bath pH increases the amplitude of native and heterologously expressed ICLH-3b only approximately twofold (9).
Kinetics of inhibition by Zn2+ are altered in CLH-3bC.
ICLH-3b in the C. elegans oocyte is inhibited by >90% by bath application of 10 mM Zn2+ (27). Because ICLH-3b and ICLH-3bC exhibit different sensitivities to extracellular Cl– and pH, we tested whether they also differ in their sensitivity to extracellular Zn2+. Oocytes were held at 0 mV and stepped to –100 mV every 2 s for 1 s during continuous perfusion of control bath or bath containing 5 mM Zn2+. Figure 7, A and C, shows typical time course experiments for Zn2+-induced inhibition of ICLH-3b and ICLH-3bC, respectively. Zn2+ inhibited ICLH-3b with a biphasic time course that consisted of a rapid initial phase followed by a much slower secondary phase (Fig. 7A). The time course of Zn2+ inhibition of ICLH-3b could be fitted with a biexponential function describing fast and slow (Fig. 7B). Mean ± SE amplitudes of the fast (Afast) and slow (Aslow) components were 0.69 ± 0.05 and 0.27 ± 0.04 (n = 4), respectively. In contrast, Zn2+ inhibited ICLH-3bC with a rapid monoexponential time course that lacked the slower phase observed during inhibition of ICLH-3b (compare Fig. 7, A and C). The time constant for Zn2+-mediated inhibition of ICLH-3bC is shown in Fig. 7D. Mean ± SE amplitude was 0.90 ± 0.01 (n = 7). Mean ± SE inhibition of ICLH-3b and ICLH-3bC with 5 mM Zn2+ at –100 mV was 86 ± 2% (n = 4) and 90 ± 1% (n = 7), respectively. The extent of inhibition in the two channels was not significantly different (P > 0.07). Block of both ICLH-3b and ICLH-3bC was reversible, and Zn2+ washout followed similar monoexponential time courses that were not significantly different for the two channels (P > 0.6) (Fig. 7, B and D). Mean ± SE amplitudes of current recovery for ICLH-3b and ICLH-3bC were 0.11 ± 0.01 (n = 4) and 0.1 ± 0.03 (n = 3), respectively.
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C is blocked by Zn2+.
In addition to observing inhibition of total channel activity, we also found that Zn2+ blocked predepolarization-induced potentiation of ICLH-3bC. Figure 8A shows mean current traces from three clh-3(ok768) oocytes recorded in control bath and in the presence of 100 µM Zn2+, which blocks prepotentiation completely. A dose-response curve for Zn2+-mediated inhibition of prepotentiation is shown in Fig. 8B. IC50 for Zn2+ inhibition of ICLH-3bC prepotentiation was 10 µM. Interestingly, Zn2+ concentrations up to 250 µM failed to block hyperpolarization-activated ICLH-3bC. Mean ± SE steady-state ICLH-3bC amplitudes recorded at –100 mV in control saline and in the presence of 250 µM Zn2+ were –21 ± 8 pA/pF and –21 ± 9 pA/pF (n = 3), respectively. These values were not significantly different (P > 0.9).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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C recorded in the worm oocyte are summarized in Table 1. The voltage sensitivity of CLH-3a is reduced, and its activation kinetics are slower than those of CLH-3b. Unlike CLH-3a, CLH-3b exhibits no depolarization-induced potentiation behavior and exhibits greatly reduced sensitivity to extracellular pH and Cl– concentration (Table 1).
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As shown in Table 1, the characteristics of ICLH-3bC suggest that the COOH terminus plays an important role in controlling channel properties. The clh-3(ok768) allele is a 165-amino acid COOH-terminal deletion that encompasses the 101-amino acid insert between the CLH-3b CBS domains and another 64-amino acids upstream of this region that are shared by both variants (Fig. 1C). Deletion of this region increases channel pH and Cl– sensitivity (Fig. 6), reduces sensitivity to hyperpolarizing voltages and slows hyperpolarization-induced activation kinetics (Fig. 4), and induces depolarization-induced prepotentiation behavior (Fig. 5).
The properties of CLH-3bC resemble but do not fully recapitulate those of heterologously expressed CLH-3a. It is noteworthy that the stimulatory effects of bath acidification, elevated bath Cl–, and a 3-s predepolarization to 60 mV on CLH-3a are about twice those observed for CLH-3bC (Table 1). This suggests that the CLH-3a NH2 terminus may be required for full sensitivity to these parameters or that sensitivity to depolarization and extracellular ions is also modulated by the 169-amino acid extension of the CLH-3b COOH terminus. Site-directed mutagenesis and heterologous expression studies are required to fully define the role of NH2 and COOH termini in CLH-3 channel gating.
Bacterial ClC crystal structures (12, 13) have demonstrated that the last -helix, or R helix, immediately preceding the cytoplasmic COOH terminus contributes a tyrosine residue that protrudes into the channel pore and functions in Cl– coordination. Amino acid residues thought to be involved in ClC fast gating and pore Cl– binding (12, 13) are fully conserved in CLH-3a and CLH-3b. We suggested previously (9) that the differences in extracellular H+ and Cl– sensitivity of the splice variants (see Table 1) might be due to structural changes in the CLH-3b COOH terminus that secondarily alter the structure of the R helix and the accessibility and/or function of pore-associated ion-binding sites. Recent studies by Hebeisen and Fahlke (17) are consistent with this idea and have shown clearly that truncation of the COOH terminus alters the conformation of the outer vestibule of ClC-1.
We also suggested previously (9) that the effect of depolarization on channel activation could be due to effects of changes in local Cl– concentration on a fast gating mechanism. However, the data shown in Fig. 8 suggest that depolarization-induced potentiation may be mediated at least in part by a slow gating process. Extracellular Cd2+ and Zn2+ inhibit ClC channels (7, 11, 21, 31), including CLH-3b (27) (Fig. 7A). Studies of ClC-0 (7) and ClC-2 (31) have suggested that facilitation of slow gating is responsible for the inhibitory effects of these cations. In ClC-1, Zn2+ inhibition is thought to be mediated by binding of the ion to a closed state of the slow gate (11). It is conceivable that depolarization-induced opening of the CLH-3bC slow gate is responsible for potentiation. Interestingly, we found that micromolar concentrations of extracellular Zn2+ inhibit depolarization-induced potentiation with an IC50 of 10 µM (Fig. 8) but have no effect on hyperpolarization-induced current amplitude. If potentiation involves a slow gating mechanism, micromolar levels of Zn2+ may disrupt depolarization-induced conformational changes that are required for opening of the gate. Detailed analysis of CLH-3 single-channel properties is needed to fully assess how voltage, extracellular ions, and NH2-terminal and COOH-terminal splice variations modulate fast and slow gating processes.
Exposure to 5 mM Zn2+ caused a rapid and reversible 90% inhibition of both CLH-3b and CLH-3bC (Fig. 7). However, the kinetics of Zn2+ inhibition for the two channels are considerably different. Fast and slow processes mediate Zn2+ inhibition of CLH-3b (Fig. 7, A and B), whereas inhibition of CLH-3bC occurs by a rapid process that can be described by a single exponential (Fig. 7, C and D). Divalent cation inhibition of ClC-0, ClC-1, and ClC-2 is thought to be mediated in part by Zn2+ binding to specific extracellular cysteine residues. In ClC-1, for example, three cysteine residues that participate in Zn2+-dependent channel inhibition have been identified on the basis of site-directed mutagenesis studies (21). The altered Zn2+ inhibition of CLH-3bC suggests that changes in the structure of the intracellular COOH terminus may modulate the accessibility and Zn2+-binding kinetics of cysteine and/or other amino acid residues on the extracellular face of the channel.
CLH-3b is activated by oocyte swelling and meiotic cell cycle progression (27). Activation is mediated by serine/threonine dephosphorylation events (10, 28). Deletion mutation of the CLH-3b COOH terminus dramatically alters channel regulation. CLH-3bC is insensitive to oocyte shrinkage (see RESULTS) and ATP depletion (Fig. 3B). These experimental maneuvers respectively inhibit (27) and dramatically activate CLH-3b (28). In addition, CLH-3b is activated >20-fold by oocyte swelling, whereas CLH-3bC is activated only 1.2-fold (Fig. 3A). Loss of CLH-3bC volume sensitivity and activation by ATP depletion suggests that the deleted amino acids are involved in channel regulation. The deleted region contains several potential phosphorylation sites, a binding site for germinal center kinase-3, or GCK-3, which is a new member of the sterile 20 (Ste20) serine/threonine kinase superfamily that functions to inhibit CLH-3b (10), and a highly charged region that could mediate regulatory protein-protein interactions and/or regulatory interactions within the channel protein itself.
In conclusion, we have characterized a CLH-3b COOH-terminal deletion mutant in its native cellular environment, where regulatory interactions with other proteins are presumably minimally perturbed. Our results demonstrate clearly that the cytoplasmic COOH terminus plays important roles in channel gating and regulation. Site-directed mutagenesis studies are clearly warranted to begin defining the underlying structural and functional relationships of the COOH terminus. Our findings contribute to a growing body of evidence indicating that NH2- and COOH-terminal cytoplasmic domains are essential for the function of eukaryotic ClCs and likely contribute significantly to the evolution and physiological diversification of these channels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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