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

Abstract

A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Cav1.3 L-type Ca2+ channels, in particular the voltage dependence of activation (V0.5) and Ca2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Cav1.3 (Cav1.3L), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCav1.3scg). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCav1.3scg with a rat cDNA isolated from rat pancreas (rCav1.3L). When expressed in tsA-201 cells under identical experimental conditions rCav1.3L exhibited Ca2+ current properties indistinguishable from human and mouse Cav1.3L, compatible with the presence of a functional CTM. In contrast, rCav1.3scg showed gating properties similar to human short splice variants lacking a CTM. rCav1.3scg differs from rCav1.3L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCav1.3scg explained most of the functional differences to rCav1.3L. Their mutation to the corresponding residues in rCav1.3L (G244, V2075) revealed that both contributed to the more negative V0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCav1.3L. We found no evidence for transcripts containing one of the single rCav1.3scg mutations in rat superior cervical ganglion preparations. However, the rCav1.3scg variant provided interesting insight into the structural machinery involved in Cav1.3 gating.