Relevance of the D13 Region to the Function of the Skeletal Muscle Chloride Channel, ClC-1

Abstract

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (⌬D13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and ⌬D13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys 877 to Arg 894 .In mammalian skeletal muscle, the voltage-gated chloride channel, ClC-1, is responsible for the greater proportion of the resting conductance, which acts to stabilize the membrane potential against unwanted perturbations. Its structure, as predicted by hydropathy analysis (1) and in common with other members of the large ClC family, includes 13 relatively hydrophobic domains (termed D1 to D13), most of which probably span the membrane. There is good evidence, however, that D4 and D13 are located entirely extracellularly and intracellularly, respectively (2, 3), with the implication that close to half the amino acids of human ClC-1 (hClC-1) 1 must form an extensive cytoplasmic tail (Gln 574 -Leu 988 ). The D13 domain, which is conserved among all eukaryotic ClC channels (4), is situated in this tail, about two-thirds of the way to the carboxyl terminus (Leu 840 -Ile 870 ). Mutations in ClC-1 have been associated with both dominant and recessive forms of myotonia (e.g. Refs. 5 and 6) characterized by abnormal, sustained firing of action potentials that result in prolonged involuntary muscle contractions. In one (R894X) of two myotonic mutations identified in the carboxyl tail (7, 8) the final 95 amino acids of the protein (8) are lost. Functional analysis of this mutant, expressed in Xenopus oocytes, showed a large reduction in chloride currents, which could account for the myotonic symptoms (9). In similar studies (4), chloride currents were totally lost from human ClC-1 truncated at Ser 720 (G721X) and at Gln 597 ...

The Role of the Carboxyl Terminus in ClC Chloride Channel Function

“…pressing isolated carboxyl-terminal proteins rescued non-functional truncation mutants of hClC-1 in mammalian cells in agreement with earlier results using Xenopus oocytes or insect cells (13)(14)(15). Channels formed by assembly of such fragments exhibit unaltered gating and conduction properties, but there were marked differences in current amplitudes between separate sets of co-expressed fragments.…”

“…1A). The functional importance of this channel region is illustrated by various disease-causing mutations (3, 6, 11, 12) and the experimental finding that certain truncations abolish function of several ClC isoforms (13)(14)(15) (Fig. 1).…”

“…The functional importance of the carboxyl terminus is illustrated by disease-causing mutations in ClC-1, -2, -5 and ClC- Kb (3,6,11,12). Moreover, truncations removing parts of the distal cystathionine-␤-synthase (CBS) 1 domain of ClC channels were shown to abolish functional expression in heterologous systems (6,(13)(14)(15)(16). Although co-expression of the complementary carboxyl terminus restored the function of these channels, this was not the case for truncations removing carboxyl-terminal fragments containing both CBS domains (13).…”

Golgi Localization and Functionally Important Domains in the NH2 and COOH Terminus of the Yeast CLC Putative Chloride Channel Gef1p

“…This effect could be blocked by brefeldin A, suggesting (in line with this work) an effect of CBS2 on trafficking. In the baculovirus system, however, an in-frame deletion of CBS2 in ClC-1 (leaving the extreme COOH terminus intact) still gave currents, showing that CBS2 is not absolutely necessary for ClC-1 function upon overexpression (64).…”

Regulation of Albumin Endocytosis by PSD95/Dlg/ZO-1 (PDZ) Scaffolds

“…A mutant that lacked the final five amino acids (D741X) was generated using site-directed mutagenesis as described previously (38). Both plasmids were used to transform BL-21 component cells.…”