title: Alternative splicing of N- and C-termini of a C. elegans ClC channel alters gating and sensitivity to external Cl- and H+.
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.
authors.affiliation: Department of Anaesthesiology, Vanderbilt University Medical Center, T-4202 Medical Center North, Nashville, TN 37232-2520, USA.
authors.forename: Jerod
authors.initials: J
authors.lastname: Denton
authors.forename: Keith
authors.initials: K
authors.lastname: Nehrke
authors.forename: Eric
authors.initials: E
authors.lastname: Rutledge
authors.forename: Rebecca
authors.initials: R
authors.lastname: Morrison
authors.forename: Kevin
authors.initials: K
authors.lastname: Strange
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics* / Q000502:physiology; D002712:Chlorides / Q000502:physiology; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics*; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D011522:Protons
chemical_list: D029742:Caenorhabditis elegans Proteins; D018118:Chloride Channels; D002712:Chlorides; D011522:Protons; C417201:clh-3 protein, C elegans