Structural determinants of CaV1.3 L-type calcium channel gating

Abstract

Cav1.3 L-type Ca2+-channels are key signal molecules for hearing, cardiac pacemaking and neuronal excitability. We have recently discovered a C-terminal intramolecular protein-interaction in Cav1.3 α1-subunits as a gating modifier (CTM). This modulatory domain is absent in short C-terminal splice variants. Its presence shifts half maximal activation voltage (V0.5) to more positive potentials and inhibits Ca2+-dependent-inactivation (CDI). This regulation is present in long variants of human (Singh et al., 2008) and rat cDNA clones from pancreatic islets (rCav1.3pan) but not in a clone derived from superior cervical ganglion (rCav1.3scg). rCav1.3scg differs from rCav1.3pan at three amino acid (aa) positions (S244G, V1104A, A2073V), a polymethionine-stretch with two additional lysines, and one alternatively spliced locus (exon 31). We took advantage of these discrete differences to determine their role for the kinetic differences between rCav1.3scg and rCav1.3pan (in voltage-dependence of activation and CDI) by expressing channel mutants in tsA-201 cells and whole-cell patch-clamp-recordings.Two aa (S244, A2073) in rCav1.3scg explained most of the functional differences to rCav1.3pan. Mutation S244G even further enhanced CDI of rCav1.3scg and shifted its V0.5 to more positive potentials. A2073V (located within the CTM) also shifted V0.5 more positive but almost eliminated CDI. The cooperative action in the double-mutant restored gating properties (CDI, V0.5) similar to rCav1.3pan. Their effects are compatible with a recently proposed allosteric CDI mechanism implying CaM-mediated inhibition of the activation gate (Tadross and Yue, 2010). Analysis of inactivation kinetics suggest that G244 decreases the open probability (Po) of the inactivated gating mode, whereas A2073V reduces the maximal Po of rCav1.3scg as evident from increased gating currents.G244 and V2073 affect CDI through different molecular mechanisms. Together they stabilize the gating behaviour of long CaV1.3 splice variants undergoing C-terminal-modulation.Support: Austrian Science Fund (F4402, P20670, W11), University of Innsbruck