Allan H. Bretag

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl‐ concentration and pH on gating of the skeletal muscle Cl‐ channel, ClC‐1, has been assessed using the voltage‐clamp technique and the Sf‐9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady‐state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl‐ concentration. 4. A point mutation, K585E, in human ClC‐1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC‐0, alters the I‐V relationship and kinetics, but retains external Cl‐ dependence. 5. When external pH is reduced, the deactivating inward currents of ClC‐1 are diminished without change in time constants while the steady‐state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC‐1, which in some ways resembles currents due to the slow gates of ClC‐0. 8. Gating appears to be controlled by a Cl(‐)‐binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Involvement of Helices at the Dimer Interface in ClC-1 Common Gating

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.

Temperature dependence of human muscle ClC-1 chloride channel

1 In the present work we investigated the dependence on temperature of the ionic conductance and gating of human muscle ClC‐1 chloride channels, transiently expressed in human embryonic kidney (HEK 293) cells. 2 At normal pH, ClC‐1 currents deactivated at negative potentials with a double‐exponential time course. The time constants of the exponential components, corresponding to the relaxations of the fast and slow gates, were temperature dependent with Q10 values of ≈3 and ≈4, respectively. Current amplitude increased with increasing temperature with a Q10 of ≈1.6. 3 The voltage dependence of the two gating processes was shifted towards more positive potentials with increasing temperature. The half‐saturation voltage (V1/2) of the steady‐state open probability (Po) was shifted by ≈23 and ≈34 mV per 10 °C increase in temperature, for the fast and slow gate, respectively. 4 At low pH, the voltage dependence of ClC‐1 was reversed and currents were activated by hyperpolarisation with a single‐exponential time course. This type of gating in ClC‐1 resembled the slow gating of the Torpedo ClC‐0 homologue, but differed with respect to its kinetics and temperature dependence, with a Q10 of gating relaxations at negative potentials of ≈5. The Arrhenius plot of ClC‐1 conductance at low pH had a clear break point at ≈25 °C, with higher Q10 values at lower temperatures. 5 The temperature sensitivity of relaxation and open probability of the slow gate, which in both ClC‐0 and ClC‐1 controls two pores simultaneously, implies that the slow gating of ClC‐1 is mechanistically different from that of ClC‐0.

pH-Dependent Interactions of Cd2+ and a Carboxylate Blocker with the Rat ClC-1 Chloride Channel and Its R304E Mutant in the Sf-9 Insect Cell Line

1 Gating of the skeletal muscle chloride channel (ClC‐1) is sensitive to extracellular pH. In this study, whole‐cell recording of currents from wild‐type (WT) ClC‐1 and a mutant, R304E, expressed in the Sf‐9 insect cell line was used to investigate further the nature of the pH‐sensitive residues. 2 Extracellular Cd2+ produced a concentration‐dependent block of WT ClC‐1 with an IC50 of 1.0 ± 0.1 mm and a Hill coefficient of 2.0 ± 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 ± 0.1 and a Hill coefficient for proton binding of 3.0 ± 0.3. Anthracene‐9‐carboxylate (A‐9‐C) block of WT ClC‐1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 ± 0.1 and a Hill coefficient for proton binding of 1.0 ± 0.2. 3 Compared with WT ClC‐1, R304E had a lower affinity for Cd2+ (IC50, 3.0 ± 0.3 mm) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 ± 0.3. In contrast, the A‐9‐C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC‐1. 4 ClC‐1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A‐9‐C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A‐9‐C binding site to affect its characteristics, but it does alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5 The block of ClC‐1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A‐9‐C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC‐1.

Modulation of the gating of ClC-1 by S-(–) 2-(4-chlorophenoxy) propionic acid

Using whole‐cell patch‐clamping and Sf‐9 cells expressing the rat skeletal muscle chloride channel, rClC‐1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. RS‐(±) 2‐(4‐chlorophenoxy)propionic acid (RS‐(±) CPP) and its S‐(−) enantiomer produced pronounced effects on ClC‐1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration‐dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S‐(−) enantiomer respectively. R‐(+) CPP at similar concentrations had no effect on gating. RS‐(±) CPP did not block the passage of Cl− through the pore of rClC‐1. ClC‐1 is gated by Cl− binding to a site within an access channel and S‐(−) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl−. Comparison of the EC50 for RS‐(±) CPP and S‐(−) CPP indicates that R‐(+) CPP can compete with the S‐(−) enantiomer for the site but that it is without biological activity. RS‐(±) CPP produced the same effect on rClC‐1 gating when added to the interior of the cell and in the extracellular solution. S‐(−) CPP modulates the gating of ClC‐1 to decrease the membrane Cl− conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

Characteristics of skeletal muscle chloride channel CIC-1 and point mutant R304E expressed in Sf-9 insect cells

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC‐1: effects on permeation and gating

1 Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC‐1, expressed in Sf‐9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole‐cell patch‐clamp technique. 2 Bi‐ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl− (1) > benzoate (0·15) > hexanoate (0·12) > butyrate (0·09) > propionate (0·047) x≈ formate (0·046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC‐1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. 3 All anions studied when applied from outside show an apparently paradoxical voltage‐dependent block of inward currents; this voltage‐dependent block could be qualitatively described by a discrete‐state permeation model with two binding sites and three barriers. 4 Effects of the external anions with aliphatic side‐chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC‐1 gating, probably, by changing Cl− affinity to the gating site. 5 Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC‐1 gating mechanism sensitive to the internal pH.

Zinc inhibits human ClC-1 muscle chloride channel by interacting with its common gating mechanism

Transition metals block the muscle Cl− channel ClC‐1, which belongs to a large family of double‐barreled Cl− channels and transporters. In the Torpedo Cl− channel ClC‐0, Zn2+ block is closely related to the common gating mechanism that opens and closes both pores of the channel simultaneously, and the mutation C212S, which locks the common gate open, also eliminates the block. In ClC‐1, however, previous results suggested that Zn2+ block is independent of gating, and that the cysteine residues involved in Zn2+ binding are in different positions to those that confer Zn2+ sensitivity on ClC‐0. In this work, we show that Zn2+ block of ClC‐1 is faster at hyperpolarized potentials where the channel is more likely to be in the closed state. Mutation C277S, equivalent to C212S in ClC‐0, which locks the common gate in ClC‐1 open, virtually eliminates Zn2+ block. A mutation, V321A, which reduces open probability of the common gate, facilitated Zn2+ block. These results demonstrate that Zn2+ block is state dependent, acting on the common gate. The extent of the block, however, is not a simple function of the open probability of the common gate. The Q10 of ∼13 of the time course of Zn2+ block, which is significantly higher than the Q10 of common gating transitions in WT ClC‐1, suggests that Zn2+ binds to a very high temperature‐dependent low‐probability closed substate of the common gate, which has not yet been characterized in this channel.

Modification of the transient outward current of rat atrial myocytes by metabolic inhibition and oxidant stress.

1. A putative function of the transient outward current (ITO) in cardiac myocytes is to modulate the shape of the action potential and, consequently, cardiac contractility. In addition, it has been suggested that this current may help protect against arrhythmias during periods of cardiac ischaemia. In our investigation of the possible anti‐arrhythmic action of ITO, we have examined its response to metabolic inhibition and oxidant stress. 2. Whole‐cell recordings were obtained from rat atrial myocytes using standard patch‐clamp techniques. Inhibition of metabolism, using 10 mM 2‐deoxy‐D‐glucose (2‐DG) to block glycolysis with or without the addition of 2 mM cyanide to block oxidative phosphorylation, led to inhibition of ITO at a holding potential of ‐70 mV. Shifting the holding potential to ‐80 mV restored ITO, suggesting that metabolic inhibition had shifted the inactivation curve of ITO in a negative direction. 3. Quasi steady‐state inactivation curves revealed a shift in ITO inactivation induced by complete metabolic inhibition with 2‐DG and cyanide. Myocytes typically contracted shortly after the shift was observed. In the presence of Ruthenium Red, contraction was delayed and myocytes could undergo several exposures to the metabolic inhibitors, each time displaying a shift in ITO inactivation. The shifts ranged between ‐7 and ‐20 mV. 4. Recovery from inactivation was determined using a two‐pulse protocol. The time constant of recovery at a holding potential of ‐80 mV reversibly shifted from 48 +/‐ 8 to 129 +/‐ 21 ms during metabolic inhibition (n = 4). 5. The activation of ITO from a holding potential of ‐100 mV shifted in a negative direction during metabolic inhibition, from a half‐activation voltage of 0.3 +/‐ 3.0 to ‐14.7 +/‐ 2.5 mV (n = 5). Such a ‐15 mV shift increases the amplitude of ITO by approximately 30% at 0 mV. 6. A shift in ITO inactivation similar to that produced by metabolic inhibition could be shown when myocytes were subjected to oxidant stress induced by either 1 mM t‐butyl hydroperoxide (TBHP) or the photoactivation of 100 nM Rose Bengal. Furthermore, an increase in pipette concentration of free Ca2+ from 20 to 200 nM also shifted ITO inactivation in a negative direction. 7. These results raise the possibility that the rise in intracellular [Ca2+] occurring during both metabolic inhibition and oxidant stress modifies activation and inactivation of ITO.(ABSTRACT TRUNCATED AT 400 WORDS)

Movement of hClC-1 C-termini during common gating and limits on their cytoplasmic location

Functionally, the dimeric human skeletal muscle chloride channel hClC-1 is characterized by two distinctive gating processes, fast (protopore) gating and slow (common) gating. Of these, common gating is poorly understood, but extensive conformational rearrangement is suspected. To examine this possibility, we used FRET (fluorescence resonance energy transfer) and assessed the effects of manipulating the common-gating process. Closure of the common gate was accompanied by a separation of the C-termini, whereas, with opening, the C-termini approached each other more closely. These movements were considerably smaller than those seen in ClC-0. To estimate the C-terminus depth within the cytoplasm we constructed a pair of split hClC-1 fragments tagged extracellularly and intracellularly respectively. These not only combined appropriately to rescue channel function, but we detected positive FRET between them. This restricts the C-termini of hClC-1 to a position close to its membrane-resident domain. From mutants in which fast or common gating were affected, FRET revealed a close linkage between the two gating processes with the carboxyl group of Glu²³² apparently acting as the final effector for both.