Alternative splicing of N‐ and C‐termini of a C. elegans ClC channel alters gating and sensitivity to external Cl− and H+

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Abstract
CLH‐3 is a meiotic cell cycle‐regulated ClC Cl− channel that is functionally expressed in oocytes of the nematode Caenorhabditis elegans. CLH‐3a and CLH‐3b are alternatively spliced variants that have identical intramembrane regions, but which exhibit striking differences in their N‐ and C‐termini. Structural and functional studies indicate that N‐ and C‐terminal domains modulate ClC channel activity. We therefore postulated that alternative splicing of CLH‐3 would alter channel gating and physiological functions. To begin testing this hypothesis, we characterized the biophysical properties of CLH‐3a and CLH‐3b expressed heterologously in HEK293 cells. CLH‐3a activates more slowly and requires stronger hyperpolarization for activation than CLH‐3b. Depolarizing conditioning voltages dramatically increase CLH‐3a current amplitude and induce a slow inactivation process at hyperpolarized voltages, but have no significant effect on CLH‐3b activity. CLH‐3a also differs significantly in its extracellular Cl− and pH sensitivity compared to CLH‐3b. Immunofluorescence microscopy demonstrated that CLH‐3b is translationally expressed during all stages of oocyte development, and furthermore, the biophysical properties of the native oocyte Cl− current are indistinguishable from those of heterologously expressed CLH‐3b. We conclude that CLH‐3b carries the oocyte Cl− current and that the channel probably functions in nonexcitable cells to depolarize membrane potential and/or mediate net Cl− transport. The unique voltage‐dependent properties of CLH‐3a suggest that the channel may function in muscle cells and neurones to regulate membrane excitability. We suggest that alternative splicing of CLH‐3 N‐ and C‐termini modifies the functional properties of the channel by altering the accessibility and/or function of pore‐associated ion‐binding sites.