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Original Article

^a From the Centro de Investigaciones Biomédicas, Universidad de Colima, 28045 Colima, México
^b Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 México Districto Federal, Mexico
^c Centre for Drug Design and Development, University of Queensland, Saint Lucia 4072, Australia
Instituto de Fisiología Celular, UNAM, Circuito Exterior, Ciudad Universitaria, 04510 México D.F., México.Fax: 525-622-5607;

dnaranjo{at}ifcsun1.ifisiol.unam.mx

-Conotoxin-PVIIA (-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. -PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in -PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, -PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, z, is 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10⁷–10⁸ M^–1 s^–1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced -PVIIA's association rate only. Removal of internal K⁺ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the -KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K⁺ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, -PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.

Key Words: pore blockade • patch clamp • Xenopus oocyte • Conus venom • Shaker K channel

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