Structure and Sodium Channel Activity of an Excitatory I₁-Superfamily Conotoxin^,

Abstract

Conotoxin ι-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I 1 -superfamily, which contains eight cysteine residues arranged in a −C-C-CC-CC-C-C-pattern. ι-RXIA (formerly designated r11a) is one of three characterized I 1 peptides in which the third last residue is post-translationally isomerized to the D-configuration. Naturally occurring ι-RXIA with D-Phe44 is significantly more active as an excitotoxin than the L-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I 1 -superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that ι-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the D-Phe44 and L-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of ι-RXIA as a voltage-gated sodium channel; ι-RXIA is an agonist, shifting the voltage dependence of activation of mouse Na V 1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in ι-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.Through adaptive evolution, marine cone snails of the genus Conus have generated more than 70,000 different venom peptides (conopeptides) (1-3). These highly diverse peptides can be organized into several structural classes, with peptides in the same structural class generally belonging to the same gene superfamily. The peptides in a given superfamily share a highly conserved precursor signal sequence and a disulfide scaffold with a characteristic number, pattern, and pairing of cysteine residues in the mature toxin (1-3). Thus, despite hypermutation of amino acids between half-cystines, the disulfide framework remains conserved within a superfamily. In addition to the accelerated evolution of conopeptide sequences, further † This work was supported in part by N. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript molecular diversity is introduced through post-translational modifications. The occurrence of diverse modifications in Conus peptides is now well established (4) but the functional consequences of most post-translational modifications remain unknown, and mechanistic insights require detailed structure/function analyses, which have not been performed in most cases.Recently, we described the I-superfamily of Conus peptides, defined by...

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