Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn 2؉ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn 2؉ strongly depends on the external pH (pK a near 6.9), whereas the block by DEPC is rather independent of the pH in the range of 5.5 to 8.5. To identify the target sites of these reagents, we constructed a total of twelve cysteine-and/or histidine-replacement mutants, transfected tsA201 cells with them, and investigated the resulting whole-cell chloride currents. The majority of the mutants exhibited a similar sensitivity toward Zn 2؉ or DEPC as wild type (WT) channels. Block by 1 mM Zn 2؉was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn 2؉ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn 2؉ -binding.The main chloride channel of human skeletal muscle, hClC-1, 1 is a member of the ClC chloride channel family that is unrelated to any other known ion channels (1). The first membrane topology model of ClC proteins was derived from hydropathy analysis (2); several times it had to be revised on the grounds of firmer experimental evidence (3-5). Certain mutations in the human ClC-1 gene (reviewed in Ref. 6) lead to myotonia, a disease characterized by muscle stiffness. The study of such myotonia-causing mutations provided first insights into the relationships between the primary sequence and the functions of this channel (7-10). Strong evidence suggests regions between transmembrane segments D3 and the end of D5 to participate in the pore-forming structure (11). However, these results are not in agreement with the most recently postulated topology model of ClC-1 (5). Furthermore, studies of ClC-0 (reviewed in Ref. 1), a homologous chloride channel in the electric organ of Torpedo, led to the suggestion of additional protein parts being involved in forming the ion conducting pathway.ClC channels most likely consist of two subunits (12), and in the case of ClC-0, each subunit was proposed to contain a single pore (4,13,14). Recent evidence speaks against this "doublebarreled" channel model, at least in the case of ClC-1 (15). We have found earlier that the exposure of hClC-1, stably expressed in HEK-293 cells, to 1 mM Zn 2ϩ leads to a massive reduction of the conducted chloride current (16). The results were compatible with the presence of at least two extracellularly accessible zinc-binding sites and a direct effect on ion permeation, e.g. by obstruction of the pore. The histidyl-reactive diethyl pyrocarbonate (DEPC), applied from the outside, also reduced the currents through hClC-1. The aim of this study was to identify the target residues for these blockers and, thereby, to draw inferences regarding the membrane topology and the potential location of pore-forming structures. It is known from other proteins, that Zn 2ϩ binding sites are of...

Mutations in the voltage-dependent skeletal muscle chloride channel, ClC-1, result in dominant or recessive myotonia congenita. The Q552R mutation causes a variant of dominant myotonia with a milder phenotype, myotonia levior. To characterise the functional properties of this mutation, homodimeric mutant and heterodimeric wild-type (WT) mutant channels were expressed in tsA201 cells and studied using the whole-cell recording technique. Q552R ClC-1 mutants formed functional channels with normal ion conduction but altered gating properties. Mutant channels were activated by membrane depolarisation, with a voltage dependence of activation that was shifted by more than +90 mV compared to WT channels. Q552R channels were also activated by hyperpolarisation, and this process was dependent upon the intracellular chloride concentration ([Cl _ ] i ). Together, these alterations resulted in a substantial reduction in the open probability at _85 mV at a physiological [Cl _ ] i . Heterodimeric WT-Q552R channels did not exhibit hyperpolarisation-activated gating transitions. As was the case for WT channels, activation occurred upon depolarisation, but the activation curve was shifted by 28 mV to more positive potentials. The functional properties of heterodimeric channels suggest a weakly dominant effect, a finding that correlates with the inheritance pattern and symptom profile of myotonia levior.

To investigate the membrane topology of the skeletal muscle chloride channel ClC-1, we inserted the small antigenic flag (DYKDDDDK) and/or HSV (QPELAPEDPED) epitope tags into nine predicted extra-and intracellular loops along the channel protein.Functional integrity of the modified proteins was tested by measuring the chloride currents conducted by these channels expressed in tsA201 cells. Insertion of the tags into the linkers D1D2, D4D5, D6D7, D8D9 or D11D12 did not alter channel function significantly, whereas insertion into D3D4, D5D6, D9D10 and D10D11 led to loss of function. Intra-or extracellular localisation of the tags was determined by immunofluorescent staining of intact and permeabilised tsA201 cells transiently transfected with the functional epitope-inserted constructs. Intact cells stained for the epitope tags inserted into D1D2, D6D7 and D8D9, indicating that these linkers face the extracellular side of the membrane. No conclusions could be drawn for the location of D4D5 and D11D12. Insertion of the flag epitope at position P260 (linker D4D5), a putative pore-lining region, did not change any of the channel function properties markedly, suggesting that the region surrounding P260 cannot directly line the ion conduction pathway of ClC-1.

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