HUMAN NEURONAL VOLTAGE-DEPENDENT CALCIUM CHANNELS - STUDIES ON SUBUNIT STRUCTURE AND ROLE IN CHANNEL ASSEMBLY

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha1, alpha2 and beta subunits. Previously, we cloned and expressed human neuronal alpha1, alpha2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha1Balpha2bbeta1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha1B subunit. Co-expression of either alpha2b or beta1 subunits with an alpha1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha2b and beta1 subunits were co-expressed with an alpha1B subunit. The presence of alpha2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha1, alpha2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.