title: Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.
abstract: Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-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 alpha 2 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 alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b 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 alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.
authors.affiliation: SIBIA, Inc., La Jolla, CA 92037.
authors.forename: P F
authors.initials: PF
authors.lastname: Brust
authors.forename: S
authors.initials: S
authors.lastname: Simerson
authors.forename: A F
authors.initials: AF
authors.lastname: McCue
authors.forename: C R
authors.initials: CR
authors.lastname: Deal
authors.forename: S
authors.initials: S
authors.lastname: Schoonmaker
authors.forename: M E
authors.initials: ME
authors.lastname: Williams
authors.forename: G
authors.initials: G
authors.lastname: Veliçelebi
authors.forename: E C
authors.initials: EC
authors.lastname: Johnson
authors.forename: M M
authors.initials: MM
authors.lastname: Harpold
authors.forename: S B
authors.initials: SB
authors.lastname: Ellis
mesh_terms: D000595:Amino Acid Sequence; D001464:Barium / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004594:Electrophysiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D020866:omega-Conotoxin GVIA
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010455:Peptides; D012333:RNA, Messenger; D001464:Barium; D020866:omega-Conotoxin GVIA