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First published online on October 17, 2003.
 Copyright © 2003 by The Physiological Society
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Received August 8, 2003
Revised September 4, 2003
Accepted after revision October 15, 2003

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

Jerod Denton1*, Keith Nehrke2, Eric Rutledge3, Rebecca Morrison1, and Kevin Strange1

1 Vanderbilt University Medical Center
2 University of Rochester Medical Center
3 Skidmore College

* To whom correspondence should be addressed. E-mail: jerod.denton{at}vanderbilt.edu.

CLH-3 is a meiotic cell cycle-regulated ClC Cl- channel functionally expressed in oocytes of the nematode C. elegans. CLH-3a and CLH-3b are alternatively spliced variants that have identical intramembrane regions, but 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 HEK-293 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. 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 likely functions in non-excitable 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 neurons to regulate membrane excitability. We suggest that alternative splicing of CLH-3 N- and C- termini modifies channel functional properties by altering the accessibility and/or function of pore- associated ion-binding sites.


Key words: Anion channel • Cl- current




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