Structure and Functional Expression of an ω-Conotoxin-Sensitive Human N-Type Calcium Channel

TLDR

N‑type calcium channels are voltage‑dependent, ω‑conotoxin‑sensitive ion channels that regulate neurotransmitter release and are formed by α1 subunits that constitute the pore. The primary structures of human neuronal calcium channel α1B subunits were determined by sequencing overlapping complementary DNAs. Two α1B splice variants (α1B‑1 and α1B‑2) were found in neuroblastoma cells and CNS but not in muscle or aorta; α1B‑1, when co‑expressed with β2 and α2b subunits in HEK293 cells, produced functional N‑type channels that were irreversibly blocked by ω‑CgTx (Kd ≈ 55 pM) and insensitive to dihydropyridines, with β2 required for activity and α2b modulating expression, indicating that subunit heterogeneity generates multiple biophysically distinct N‑type channels.

Abstract

N-type calcium channels are ω-conotoxin (ω-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the α 1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel α 1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (α 1B-1 and α 1B-2 ) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The α 1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal β 2 and α 2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by ω-CgTx but was insensitive to dihydropyridines. The α 1B-1 α 2b β 2 -transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) ω-CgTx binding sites. Co-expression of the β 2 subunit was necessary for N-type channel activity, whereas the α 2b subunit appeared to modulate the expression of the channel. The heterogeneity of α 1b subunits, along with the heterogeneity of α 2 and β subunits, is consistent with multiple, biophysically distinct N-type calcium channels.