Identification of Three Cysteines as Targets for the Zn2+ Blockade of the Human Skeletal Muscle Chloride Channel

Abstract

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...

Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH

“…For example, DEPC inhibited NHE1 activity (42) and aquaporin 1-mediated water transport (33,34) in Xenopus oocytes at pH o 6.0. In contrast, DEPC inhibition of the ClC-1 Cl Ϫ channel was pH o independent between pH o 5.5 and 8.5 (22).…”

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte

“…Divalent cation inhibition of ClC-0, ClC-1, and ClC-2 is thought to be mediated in part by Zn 2ϩ binding to specific extracellular cysteine residues. In ClC-1, for example, three cysteine residues that participate in Zn 2ϩ -dependent channel inhibition have been identified on the basis of site-directed mutagenesis studies (21). The altered Zn 2ϩ inhibition of CLH-3b⌬C suggests that changes in the structure of the intracellular COOH terminus may modulate the accessibility and Zn 2ϩ -binding kinetics of cysteine and/or other amino acid residues on the extracellular face of the channel.…”

“…8 suggest that depolarizationinduced potentiation may be mediated at least in part by a slow gating process. Extracellular Cd 2ϩ and Zn 2ϩ inhibit ClC channels (7,11,21,31), including CLH-3b (27) (Fig. 7A).…”

Inhibitory Mechanism of Store-operated Ca2+ Channels by Zinc

“…The properties of the zinc inhibitory site on the SOCC and the CIC chloride channel are similar. On the latter, zinc ions interact with 3 cysteines on the extracellular site of the ion permeation pathway (35). It will be interesting to determine whether there are common structural motives on CIC channel and Trps, related to the zinc inhibitory site.…”

“…On other proteins such as the glycine receptor, CIC chloride channel, and the L-type Ca 2ϩ channel, zinc acts as a competitive inhibitor of ion permeation (33)(34)(35). To distinguish between these two modes of inhibition, the K i for Zn 2ϩ inhibition of Ca 2ϩ influx through the SOCC was plotted versus extracellular Ca 2ϩ concentration (Fig.…”