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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.

Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn2+ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn2+ strongly depends on the external pH (pKa 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 Zn2+ or DEPC as wild type (WT) channels. Block by 1 mM Zn2+ was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn2+ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn2+-binding.