Abstract

kappa-Conotoxin-pviia (K-pviia) is derived from a hunting marine snail and belongs to a family of peptides specific for a wide variety of ion channels and receptors. K-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 K-pviia can exhibit strong structural mimicry to that of charybdotoxin, an a-KTX scorpion toxin that occludes the pore of K+ channels (MacKinnon & Miller, 1 988; Scanlon et al. 1997) Using the well-known a-KTX inhibition mechanism on K+ channels as a paradigm for comparison, we studied the mechanism by which K-pviia inhibits Shaker K + channels expressed in Xenopus oocytes, with the N -type inactivation removed (Garcia et al. 1999). K-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 t o open channels in outside-out membrane patches, indicating that the toxin binds with similar, although not identical, affinities to open and closed states. The effective valence of the voltage dependence, z8, is ~0·55 and resides 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 only the K-pviia association rate. A further t est of the pore occlusion mechanism was performed by complete replacement of the internal K+ with N-methyl-D-glucamine. This experimental manoeuvre produced two main effects: (a) at any explored voltage it reduced the dissociation rate by 50 % or more , and (b) it reduced z8 of the dissociation rate to a value < 0'3. This trans-pore effect of the internal K + removal suggests that (a) as in the a -KTX, a positively charged side chain of the toxin interacts, perhaps electrostatically, with ions residing inside the Shaker pore; and (b) a part of the toxin protrudes into the pore occupying an externally accessible binding site that is usually occupied by K+ in the unblocked channel, thus decreasing its degree of occupancy by permeant ions. Occupation of this external binding site by permeant ions impeded or delayed C-or P-type inactivation, with pore-collapsing conformational changes occurring near the external entrance of the Shaker pore (Lopez-Barneo et al. 1993). Thus, we tested whether we could promote any of these non-conducting states just by blocking the channels with K-pviia in the absence of internal pel'meant ions to empty the pore. We found that the toxin prevents the channel entering into C-or P -ty pe inactivation states, even if the pore is putatively empty of permeant Ions . Although evolutionarily distant to a-KTX scorpion toxins, K-pviia shares their mechanism of inhibition of K+ channels. Two characteristics of K+ channel peptide toxins also suggest structural convergence: (a) toxin affinity for its receptor is disrupted mostly by mutations in a lysine and in an aromatic residue that are usually ~0'7 nm apart, suggesting a functional dyad (Dauplais et al. 1997), and (b) they have an excess of positive charges distributed in a manner that generates a dipole moment oriented perpendicular to the interaction surface of the toxin. This dipole may play an important role in the overall probability of colliding with a negatively charged vestibule in the proper orientation (Garcia et at. 1999). Thus , some peptide toxins seem to have adopted a common mechanism of action on K+ channels; they bind with 1: 1 stoichiometry to its most conserved structural locus, the pore . Such a precise mechanism appears to constrain the possible ways in which K+ channel-specific peptide toxin can be structured. Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, E., Roumestand, C., Vita, C., de Medeiros, C.L.C., Rowan, E.G., Harvey, A.L. & Menez, A. (1997). J. Bioi. Chem. 272, 4302- 4309. Garcia, E., Scanlon, M. & Naranjo, D. (1999). J. Gen. Physiol. 114, 141-157. Lopez-Earneo, J. , Hoshi, T., Heinemann, S. & Aldrich, R. (1993). Recept. Chann. 1, 61-7l. MacKinnon , R. & Miller, C. (1988). J. Gen. Physiol. 91,335-349. Scanlon, M., Naranjo , D., Thomas, L., Alewood, P., Lewis, R. & Craik, D (1997). Structure 5,1585-1597