Dependence of μ-conotoxin block of sodium channels on ionic strength but not on the permeating [Na+] -: Implications for the distinctive mechanistic interactions between Na+ and K+ channel pore-blocking toxins and their molecular targets

被引:10
作者
Li, RA
Hui, K
French, RJ
Sato, K
Henrikson, CA
Tomaselli, GF
Marbán, E
机构
[1] Johns Hopkins Univ, Sch Med, Inst Mol Cardiobiol, Baltimore, MD 21205 USA
[2] Univ Calgary, Dept Physiol & Biophys, Calgary, AB T2N 4N1, Canada
[3] Fukuoka Womens Univ, Fukuoka 8138529, Japan
关键词
D O I
10.1074/jbc.M301039200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
mu-Conotoxins (mu-CTXs) are Na(+) channel-blocking, 22-amino acid peptides produced by the sea snail Conus geographus. Although K(+) channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (mu), no such dependence has been reported for mu-CTX and Na(+) channels. Such properties would offer insight into the binding and blocking mechanism of mu-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of mu and permeant ion concentration ([Na(+)]) on mu-CTX block of rat skeletal muscle (mu1, Na(v)1.4) Na(+) channels. mu-CTX sensitivity of wild-type and E758Q channels increased significantly (by similar to20-fold) when mu was lowered by substituting external Na(+) with equimolar sucrose (from 140 to 35 mM Na(+)); however, toxin block was unaltered (p > 0.05) when mu was maintained by replacement of [Na(+)] with N-methyl-D-glucamine (NMG(+)), suggesting that the enhanced sensitivity at low mu was not due to reduction in [Na(+)]. Single-channel recordings identified the association rate constant, k(on), as the primary determinant of the changes in affinity (k(on) increased 40- and 333-fold for mu-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mM Na(+) was reduced to 50 mM). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional mu-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity external mu. Taken together, our findings indicate that mu-CTX block of Na(+) channels depends critically on mu but not specifically on [Na(+)], contrasting with the known behavior of pore-blocking K(+) channel toxins. These findings suggest that different degrees of ion interaction, underlying the fundamental conduction mechanisms of Na(+) and K(+) channels, are mirrored in ion interactions with pore-blocking toxins.
引用
收藏
页码:30912 / 30919
页数:8
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