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Original Article |
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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