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Electrophysiological Characteristics of Six Mutations in hClC-1 of Korean Patients with Myotonia Congenita

ClC-1 is a member of a large family of voltage-gated chloride channels, abundantly expressed in human skeletal muscle. Mutations in ClC-1 are associated with myotonia congenita (MC) and result in loss of regulation of membrane excitability in skeletal muscle. We studied the electrophysiological characteristics of six mutants found among Korean MC patients, using patch clamp methods in HEK293 cells. Here, we found that the autosomal dominant mutants S189C and P480S displayed reduced chloride conductances compared to WT. Autosomal recessive mutant M128I did not show a typical rapid deactivation of Cl− currents. While sporadic mutant G523D displayed sustained activation of Cl− currents in the whole cell traces, the other sporadic mutants, M373L and M609K, demonstrated rapid deactivations. V1/2 of these mutants was shifted to more depolarizing potentials. In order to identify potential effects on gating processes, slow and fast gating was analyzed for each mutant. We show that slow gating of the mutants tends to be shifted toward more positive potentials in comparison to WT. Collectively, these six mutants found among Korean patients demonstrated modifications of channel gating behaviors and reduced chloride conductances that likely contribute to the physiologic changes of MC.

. Two types of myotonia are recognized: the autosomal dominant disease called myotonia congenita (Thomsen's disease, OMIM 160800) (George et al., 1993) and the autosomal recessive disease called generalized myotonia (Becker's disease, OMIM 255700) (Hsiao et al., 2010;Jentsch et al., 2010;Koch et al., 1992;. Both conditions display similar clinical features, but can be distinguished based on severity and inheritance patterns (Koch et al., 1992;. For example, it is commonly reported that the recessive pattern of the disease is more severe than the dominant pattern (Koch et al., 1992;Lossin and George, 2008). However, the causative factors of either pattern of disease have yet to be uncovered (Lossin and George, 2008;Tang and Chen, 2011). It has been known that mutations in the gene encoding the skeletal muscle Clchannel, ClC-1 are a major contributor to the pathogenesis of MC (Lossin and George, 2008;Tang and Chen, 2011).
ClC-1 is one of a subfamily of voltage-gated anion channels, and is distributed mainly to T-tubules and sarcolemmal membranes in skeletal muscle (Bryant et al., 1971;Dulhunty, 1979;Fahlke et al., 1997a;Heiny et al., 1990). ClC-1 plays a significant role in stabilization of the membrane potential at the resting level after action potential firing from influx of sodium ions through voltage-gated sodium channels (Bryant et al., 1971;Dulhunty, 1979;Fahlke et al., 1997a). Structurally ClC-1 protein forms a homodimeric double-barrel and consists of 18 domains and two tandem cystathionine-β-synthase (CBS) domains located in the intracellular c-terminal region (Markovic and Dutzler, 2007;Meyer and Dutzler, 2006). The CBS domain is one of the key structures observed in the eukaryotic ClC family. Two CBS domains are located in the cytoplasmic c-terminal and are essential to ClC channel function (Dutzler et al., 2002;Markovic and Dutzler, 2007;Meyer and Dutzler, 2006).
Cell culture and transient transfection HEK293 cells (American Type Culture Collection, USA) were incubated in Dulbecco's modified Eagle's medium (Hyclone), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ug/ml streptomycin according to the supplier's recommendations. hClC-1 and mutant channels were transiently expressed using fugene 6 transfection reagent (Promega), based on manufacturer's guide. To mimic heterozygous effect of autosomal dominant pattern, mutants were co-expressed in 1:1 ratio with WT, using fugene 6 transfection reagent (Promega) and incubated in 37°C. All experiments were conducted after 24-48 h of transfection.
Construction of DNA plasmids pEGFP-N1 (Clontech) was substituted for the mammalian expression vector construct pRc/CMV (Invitrogen) containing hClC-1. The restriction enzyme sites were XhoΙ and EcoRΙ. We utilized site-directed mutagenesis with QuikChange XL Site-Directed Mutagenesis Kit.
The 12 voltage pulses for the experiment were applied from -165 mV to 75 mV in 20 mV increments. Each voltage pulse was given with a 160 ms duration. For S189C, the voltage pulses were added up to 195 mV to obtain a complete Boltzmann fitting. Holding potential was fixed at 0 mV, and tail pulse was given at -125 mV for 20 ms duration in the voltage pulse protocol. For the low chloride experiment, holding potential was fixed at -85 mV.
Open probability of slow gating was determined from a step pulse protocol similar to those for overall P o , with the addition of a short (500µs) prepulse to 170 mV between a series of step pulses and -125 mV tail pulse, to maximize P o of fast gating (Accardi and Puschi, 2000).

Data analysis
Data analysis was conducted using Origin 8 (OriginLab) and Clampfit (Axon Instruments). Overall apparent open probability (P o ) for WT and six mutant channels was calculated from the tail currents after voltage pulses ranging from -164 mV to 75 or 195 mV.
where P min is an offset, or a minimum P o at the most negative potential, V is the membrane potential, V 1/2 is the half-maximal activation potential, and κ is the slope factor.
For separation of slow and fast gating, where P s o and P f o indicate the open probability of slow and fast gating at V p . N is the number of channels expressed at -120 mV. Here, when prepulse at +170 mV is given after a series of voltage steps, which can be assumed that the open probability of fast gating is ~1. In this state, the slow gate is not substantially affected. Therefore, the equation, is established. When P s o (V p ) was obtained, using the equation (3), can be drawn. Data obtained using the equation (5) were plotted. Peak current density was calculated for comparison of data Dashed lines indicate zero current line (top). Currents were measured initially (black), after steady-state inhibition in 50 μM and 500 μM DIDS for 30 s (red). Reduced maximal currents obtained at 75 mV under 50 μM (n = 5) and 500 μM (n = 5) DIDS treatment compared to initial currents were indicated in the bar graph (bottom). across cells of different sizes. It was obtained from peak current (pA) from each recording divided by cell capacitance (pF).
The time course of current activation of S189C was examined by fitting the currents obtained in 134 mM [Cl -] i between 35 mV and 115 mV in 20 mV increments.
where τ is the time constant of current activation, and A, I 0 , I (t) are the amplitudes, the offset, and the normalized currents, respectively.

Statistical analysis
Experimental values were analyzed for mean and s.e.m. The quantitative figures in this work represent the mean ± s.e.m. Data sets were statistically evaluated utilizing an unpaired t-test. Differences in the mean values of P < 0.05 were considered significant unless indicated otherwise.

Channel characteristics of WT hClC-1
Whole cell patch-clamp recordings from HEK 293 cells expressing WT hClC-1 were performed in both 134 mM [Cl -] i and 4 mM [Cl -] i to mimic physiological condition. WT hClC-1 demonstrated an instantaneous activation at the tested voltage ranges and a typical rapid deactivation of Clcurrents over time at the hyperpolarized regions after instantaneous increase under 134 mM [Cl -] i . (Fig. 1Aa). Under physiological condition (4 mM), WT hClC-1 generated instantaneous currents and slow activation of Clcurrents on a typical time course (Fig. 1Ab). To exclude the possibility of endogenous channels influencing the chloride currents, mock transfection test was performed with HEK cells expressing pEGFP-N1 vector alone under 134 mM [Cl -] i , and no currents were observed. (Fig. 1Ac). Instantaneous and steady-state current densities (pA/pF) were obtained from the representative current traces under both conditions to observe the current-voltage relationships. For instantaneous currents, the inward rectifications were observed in 134 mM [Cl -] i ] i in HEK293 cell expressing hClC-1. For voltage protocol, each holding potential and tail pulse was fixed at 0 mV and -125 mV. A series of voltage step pulses was applied from -165 mV to +75 mV in 20 mV steps for WT or mock transfection test. b) Representative whole cell current traces for WT hClC-1 and voltage protocol for hClC-1 channel activity under 4 mM [Cl -] i in HEK293 cell expressing hClC-1. For voltage protocol, each holding potential and tail pulse was fixed at -85 mV and -125 mV. A series of voltage step pulses was applied from -165 mV to +75 mV in 20 mV steps. c) Mock transfection test in HEK 293 cells. pEGFP-N1 vector was transiently expressed in HEK293

A B
were fit with a Boltzmann distribution, and V 1/2 parameters are reported in Table 2. Dashed lines indicate zero-current level.
( Fig. 1Ad). For steady-state currents, the current-voltage relationship showed an outward rectification under physiological condition (4 mM) (Fig. 1Ae). Current-voltage relationships of WT hClC-1 under 4 mM [Cl -] i revealed that reversal (equilibrium) potentials were shifted to -60 mV (instantaneous currents) and -45 mV (steady-state currents), respectively. The reversal potential was shifted to lesser degree than expected. The discrepancy may be due to permeability when glutamate used as a substitute for internal Cl -. Western blotting analysis was conducted to evaluate protein expression. Expression of WT and mutant proteins was confirmed in HEK 293 cells over expressing GFP-tagged WT hClC-1 and mutant hClC-1 proteins (Fig. 1B). Chloride channel expression was also evaluated using the general chloride channel blocker, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), as previously described (Matulef et al., 2008). When extracellular 50 μM DIDS was applied, Clcurrents were only minimally blocked (5%). However, application of extracellular 500 μM DIDS caused Clcurrents to be inhibited up to 25% compared to initial currents, and this result was similar to that previously reported (Matulef et al., 2008) (Fig. 1C). Taken to-gether, these results suggest that WT hClC-1 and the mutants were properly expressed, and the characteristics of WT hClC-1 were confirmed.

Channel characteristics of autosomal dominant mutants, S189C and P480S
Whole cell patch-clamp recording from HEK293 cells expressing WT CIC-1 showed typical voltage-dependent Clcurrents with V 1/2 of -54 ± 3 mV (Fig. 2Ac, Table 2). Surprisingly, the channel activation of the S189C mutant showed more than a 100 mV shift of V 1/2 value toward depolarizing voltage (58 ± 4 mV) with shallower voltage-dependency ( Fig. 2Ac and Table 2). In order to clarify the type of gating process involved in the phenotype of S189C mutant, whole cell recordings were performed using the "umbrella protocol" described earlier (Accardi and Pusch, 2000). V 1/2 values of WT for "slow" gating and "fast" gating were -36 ± 17 mV and -60 ± 10 mV, respectively (Table  3), well matched with the previously reported values (Accardi and Pusch, 2000). For S189C mutant, V 1/2 of both gating processes was shifted toward more depolarizing potentials relative to WT (Fig. 3Bb). In particular, the V 1/2 of slow gating was re- Whole cell current traces for S189C and P480S. For voltage protocol, a series of voltage step pulses was applied from -165 mV to 195 mV (S189C) and 75 mV (P480S). Each holding potential and tail pulse was fixed at 0 mV and -125 mV. b) Enlargement of whole cell current traces elicited during tail pulse for S189C and P480S. c) The voltage dependence of activation of WT, S189C, and P480S, calculated from tail currents measured at -125 mV. Closed black squares and opened red squares represent WT (n = 10) and mutants, S189C (n =10), P480S (n = 14), respectively. The averaged data A B C tion, and V 1/2 parameters are reported in Table 3 markably shifted by 86 mV, implying that altered "slow" gating process is mainly responsible for the functional consequence of the S189C mutation (Table 3). This right shifted voltagedependency also resulted in remarkably decreased Clcurrent density (~5% of WT channel) in the range of physiological membrane potential (Table 1). One interesting feature of S189C is slow activation as compared to instantaneous activation of WT hClC-1. The activations at the depolarized regions were voltage dependent ( Fig. 4 and Table 5).
Interestingly, P480S showed largely unaffected characteristics of both slow and fast gating processes as compared to WT ( Fig. 2Bc and Fig. 3Cb). Though it is difficult to explain the abnormal phenotype of P480S mutation given this WT-like vol-tage-dependency, the P480S mutation might lead to reduced channel activity in the plasma membrane. The decreased Clconductance (~26% of WT channel) from P480S could support this possiblity (Table 1).

Co-transfection experiment for two AD mutants
Illustrating the heterozygous form of autosomal dominant mutants, co-expression experiments were conducted in order to recapitulate heterozygous effects. Interestingly, when coexpressed with WT in HEK293 cells, S189C showed both inwardly and outwardly rectifying currents in whole cell traces (Fig. 5A). In the channel activation of co-expressed WT with S189C, V 1/2 of slow and fast gating were 45 ± 5 mV and -37 ± 8 Holding potential and tail pulse were fixed at 0 mV and -125 mV. First series of voltage pulses was given from -165 mV to +75 mV during 160 ms, and prepulse at +170 mV during 0.5 ms was followed. Second series of voltage pulses was followed after previous pulse, and it is identical to the first voltage pulse protocol except prepulse. b) Whole cell current traces of hClC-1 WT elicited from the voltage pulse protocol for fast and slow gating analysis. c) Whole cell tail current traces at -125 mV. d) The voltage dependence of activation of WT for slow and fast gating analysis, calculated from tail currents measured at -125 mV. Black closed squares and red opened squares represent relative open probability of fast (n = 10) and slow (n = 10) gating, respectively. The averaged data were fit with a Boltzmann distribu-  mV, respectively (Table 4). For S189C co-expressed with WT, slow gating process was significantly shifted more than 100 mV, suggesting that slow gating still governs the function of the channel. Co-expression tests of P480S also suggest that altered slow gating plays a key role in the functional consequence of the channel. V 1/2 of co-expressed P480S in slow gating clearly showed less voltage dependency relative to WT whereas fast gating was largely unaffected (-50 ± 5 mV) (Fig.  5Cb). Interestingly, compared to V 1/2 of S189C and P480S, V 1/2 of both AD mutants co-expressed with WT displayed less shifting toward depolarizing potentials (Tables 3 and 4). These results show that 1) co-expression experiments with WT mimic dominant negative effects, and 2) the slow gating process largely contributes to the function of the channel.

Unique behaviors of autosomal recessive mutant channel
M128I is the only autosomal recessive mutant among these six mutants. Surprisingly, M128I did not show the typical deactivation of Clcurrent on a time course (Fig. 6Aa). In addition, the channel activation of M128I was consistent with open state of M128I (Fig. 6Ac), indicating drastically right shifted V 1/2 toward more depolarizing potentials by 100 mV (56 ± 2 mV) ( Table 2). In slow gating analysis, however, M128I displayed reversed voltage-dependency (Fig. 6Bb). V 1/2 of fast gating in M128I was shifted to more depolarizing potentials by 30 mV relative to WT (Table 3). Collectively, this modified voltage-dependency in both gating is responsible for nearly 72% reduction in Clcurrent density (-138.5 ± 12.4 pA/pF) relative to WT (Table 1). The remarkably reduced Clcurrent density and the right shifted V 1/2 in fast gating can still explain the pathological output of M128I mutation in MC patient.

Various characteristics of sporadic mutants
In addition to the inherited mutants, some mutants are not as-

A B
C sociated with a familial history. M373L, G523D, and M609K are sporadic mutants, and whole cell patch-clamp recording from HEK 293 cells described the characteristics of these three mutants. M373L and M609K showed similar behaviors as WT, illustrating rapid activation and slow deactivation of Clcurrents on a typical time course (Figs. 7A and 7C). However, deactivation of current was completely absent in G523D (Fig. 7Ba). The reversed channel activation was observed in G523D whereas M373L and M609K showed WT-like characteristics (Fig. 7Bc). Interestingly, all sporadic mutants commonly revealed the reversed voltage dependency in slow gating process (Fig. 8Ab, Bb, and Cb). V 1/2 values of M373L and M609K for fast gating were -36 ± 3 mV and -50 ± 4 mV, respectively, suggesting there was no significant effect on fast gating process in the two mutants (Table 3). However, M609K resulted in a decreased Clconductance (~30% of WT channel), suggesting destabilization of the membrane potential. Interestingly, G523D showed mainly characteristics in fast gating when compared to the WT channel (Fig. 8Bb). The typical voltage dependency in the channel activation was totally abrogated, and fitting with Boltzmann distributions was imposs-  Closed black squares and opened red squares represent WT (n = 10) and mutants, WT and S189C (n = 5), WT and P480S (n = 7), respectively. The averaged data were fit with a Boltzmann distribution, and V 1/2 parameters are reported in Table 3.  (n=10), respectively. The averaged data were fit with a Boltzmann distribution, and V 1/2 parameters are reported in Table 3. For voltage protocol, a series of voltage step pulses was applied from -165 mV to 75 mV. Each holding potential and tail pulse was fixed at 0 mV and -125 mV. b) Enlargement of whole cell current traces elicited during tail pulse for M128I. c) The voltage dependence of activation of M128I, calculated from tail currents measured at -125 mV. Closed black squares and opened red squares represent WT (n=10) and M128I (n=12), respectively. The averaged data were fit with a Boltzmann distribution, and V 1/2 parameters are reported in Table 2 ]i. For voltage protocol, a series of voltage step pulses was applied from -165 mV to 75 mV. Each holding potential and tail pulse was fixed at 0 mV and -125 mV. b) Enlargement of whole cell current traces elicited during tail pulse for M373L, G523D, and M609K. Dashed line indicates zero current level. c) The voltage dependence of activation of M373L, G523D, and M609K, calculated from tail currents measured at -125 mV. Closed black squares and opened red squares represent WT (n = 10) and mutants, M373L (n = 6), G523D (n = 12), M609K (n = 7), respectively. The averaged data were fit with a Boltzmann distribution, and V 1/2 parameters are reported in Table 2. Comparison of slow gating channel activation curves (left) and fast gating channel activation curves (right) of WT and sporadic mutants. Black closed squares and red opened squares represent WT (n = 10) and mutants, M373L (n = 5), G523D (n = 10), M609K (n = 8), respectively. The averaged data were fit with a Boltzmann distribution, and V 1/2 parameters are reported in Table 3.

DISCUSSION
We report the channel gating properties of six mutants found among Korean patients. The clinical presentation and inheritance associated with these mutants underscore the importance of their channel function on physiology.
We identified the functional consequences of the autosomal dominant mutants through the whole cell patch-clamp approach. Firstly, S189C demonstrated not only outwardly rectifying chloride currents but also modified channel gating in both slow and fast gating modalities. Co-expressed S189C with WT showed both inwardly and outwardly rectifying currents as well as shifted V 1/2 to more depolarizing potentials. The S189C mutation is located in the linker region between domains C and D of ClC-1, which is known for the GSGIP region, a well-conserved domain among different species, functioning as the Cl-selectivity filter (Dutzler et al., 2002). Helices D, F, N, and R participate in the formation of the chloride conducting pore of a ClC monomer (Dutzler et al., 2002). The Clselectivity may be destabilized due to the mutation that occurs at this position. Taking into consideration both the importance of conserved residues and significant alteration that occurred in channel gating, we hypothesize that mutant S189C specifically plays a key role in slow gating of ClC-1 channel, causing outwardly rectifying currents. At depolarizing potentials, S189C activated with time constants that were drastically increased compared to WT which cannot be analyzed due to simultaneous and rapid deactivation of current. The slow activation observed in S189C might partially cause the repolarization of the action potential, leading to myotonic runs. In contrast, mutant P480S sustained whole cell traces that are similar to WT. P480S showed an appreciable right shift in slow gating whereas fast gating was not significantly altered. Co-expression of both S189C and P480Ss resulted in a shift to more depolarizing potentials, particularly in slow gating, although less pronounced than that observed for solely mutant expressed channels. Collectively, these data suggest that a dominant negative effect is seen for both mutants, affecting slow gating in particular.
M128I resulted in significant changes not only in the whole cell current but also in gating. Although current was not deactivated on a time course as in WT, the whole cell density was reduced approximately 70%, which could explain the clinical abnormalities of MC found in the patient. Furthermore, both fast and slow gating of M128I was significantly modified. Taken together, we hypothesize that significant alteration in channel function as well as reduction in chloride conductance contributes to the clinical presentation of autosomal recessive MC. It is also interesting that M128V was previously reported to cause autosomal dominant type of MC (Grunnet et al., 2003), suggesting that the specific amino acid position mutated is not a major factor in determining the inheritance pattern. M128V resulted in a slow deactivation of outwardly rectifying current in the whole cell trace whereas it showed overlapping behavior with that of M128I, including a reduction in chloride current and right shifted V 1/2 (Grunnet et al., 2003). Thus, we conclude that substitution of isoleucine instead of valine in domain B might abolish a typical slow deactivation of Clcurrent in ClC-1. Further studies regarding structures of amino acid were not conducted in this study, but it reinforces the importance of this helix part of gating function (Grunnet et al., 2003).
In the sporadic mutants, a reduced chloride conductance in both G523D and M609K was observed. Slow gating of M373L, G523D, and M609K was commonly activated at negative potentials, which is the opposite behavior to that of WT. In addition, G523D demonstrated abnormal function in fast gating. Although the property of G523D was not investigated further in this study, it is plausible that the mutant is deeply involved in both slow and fast gating mechanisms. M609K is the only mutation in the present study that is located in the CBS1 domain ( Supplementary Fig. 1). This site could be crucial in regulation of chloride conductance and slow gating in that channel activation of fast gating in M609K was similar to WT. M373L, on the other hand, simply resulted in the modified channel activation in slow gating, although it produced sufficient chloride conductance at physiologically relevant potentials. It remains puzzling how increased Clconductance induces myotonia. Recently, Richman et al. (2012) showed that the G233S mutant results in a near constitutively open channel by rendering the fast gate of the channel mostly open, while the common gating appears to be relatively normal (Richman et al., 2012). The functional characterizations of the mutant appear to be consistent with the structural role of the G233 residue according to the highresolution structures of the homologous bacterial ClC molecules (Richman et al., 2012). This "gain-of-function" mutation for the molecular function of ClC-1 paradoxically appears to result in a reduction in membrane stability leading to myotonia. Because an increase in Clconductance in response to membrane depolarization is essential to repolarize the membrane potential to the resting state, a defect in the dynamics of the response to a voltage change as occurs in the G233S mutant may contribute to the over-excitability of the membrane and the clinical myotonia, in contrary to the previous studies (Richman et al., 2012). In this regard, the effect of the G523D mutation can be considered to be similar to those caused by the loss-offunction mutations of CLCN1 that result in reduced conductance at positive membrane potentials.
The two patients with P480S or G523D mutations responded well to mexiletine (Moon et al., 2009). The common findings between these two mutations are 1) high open probability of both slow and fast gating at the hyperpolarized membrane potentials and 2) low open probability of both slow and fast gating at the depolarized membrane potentials; although both effects are more severe with the G523D mutation. The prolonged repolarization, hyperexcitability, and spontaneous action potentials would be expected if there was a reduction in the resting muscle chloride conductance through ClC-1 (gClC-1). Our findings suggest that prolonged repolarization due to decreased P o of slow gating at the depolarized regions of ClC-1 channels is important for MC and decreasing Na + current by mexiletine improved the myotonia symptoms. Decreased Clconductance was not a common factor for the response to treatment with mexiletine, a Na + channel blocker causing a reduction in Na + current amplitude.
The response to treatment is poor for M609K and M128I but excellent for P480S and G523D (Moon et al., 2009). The main physiologic difference is the voltage sensitivity (κ) of ClC-1 channel. The lower voltage sensitivity of ClC-1 mutants P480S and G523D appears to be important for the response to treatment. A similar finding is observed in mutant S189C (κ of 59 mV). However, the mutation is associated with a fair response to treatment in our previous study (patient 6 versus patient 9) (Moon et al., 2009). Although both patients have the same mutation, the symptoms and signs such as muscle hypertrophy, transient weakness, cold sensitivity, CK level, and EMG findings, were different (Moon et al., 2009).
The clinical severity of MC may be graded by Jøegensen (Colding-Jørgense, 2005). The severity grade was 5 (pronounced myotonia, transient weakness, and dystrophic features) for M609K and 2 [mild (and/or fluctuating) symptoms] for the M128I mutation (Moon et al., 2009). Both mutants commonly displayed reduced Clconductances. The open probability of slow gating is similar, but the open probability of fast gating is quite different between two mutants. These results suggest that right shift of the voltage dependence of fast gating is important for the clinical severity if other parameters such as decreased Clconductance and changes in slow gating (a reversed relationship between P o and voltage) are similar.
In conclusion, we identified and characterized six novel mutants found in ClC-1 among Korean patients who suffer from MC. The electrophysiological property as well as analysis of slow and fast gating is expected to provide a sufficient basis for investigation of ClC-1 channels in both physiological and pathological terms.
Note: Supplementary information is available on the Molecules and Cells website (www.molcells.org).