Structural determinants of the blockade of N-type calcium channels by a peptide neurotoxin

Abstract

NEUROTOXINS that selectively block Na⁺, K⁺ or Ca²⁺ channels have provided valuable information about the functional diversity of the voltage-gated channel superfamily¹. For Ca2+ channels, a variety of toxins have been found to block individual channel types^2,3. The best-known example is ω-conotoxin-GVIA, a member of a large family of peptide toxins derived from venomous cone snails^2,3, which potently and selectively blocks N-type Ca²⁺ channels^4–9, allowing their purification^10,11, cellular localization^12,13, and the elucidation of their roles in Ca²⁺ entry¹⁴, neurotransmitter release^15,16 and neuronal migration¹⁷. In contrast to Na⁺ and K⁺ channels, little is known about the molecular features that underlie Ca²⁺-channel susceptibility to toxin block; it is also unknown whether block occurs by direct physical occlusion³ or an action on channel gating¹⁸. Here we describe structural determinants of the N-type Ca²⁺ channel's interaction with ω-conotoxin-GVIA. When chimaeras combining individual motifs from the N-type channel and from a channel insensitive to ω-conotoxin-GVIA were expressed in ^Xenopus oocytes, each of the four motifs appeared to contribute to interaction with the toxin. The most dramatic effects on toxin interactions were seen at a single cluster of residues in the large putative extracellular loopbetween IIIS5 and IIIH5, consistent with a direct pore-blocking mechanism. These results provide a starting point for delineating the architecture of the outer vestibule of the Ca²⁺ channel.