Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells

Chie Kondo,Issei Seyama,Masayuki Inoue,Hiroshi Miyoshi,Eiji Kinoshita,Kaoru Yamaoka,Keisuke Miyazaki,Masahiro Hirama

doi:10.1074/jbc.m806481200

Abstract

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

Highlights

Ciguatoxin is a lipophilic cyclic polyether with 13 ether rings (Fig. 1) derived from the dinoflagellate Gambierdiscus toxicus [1] and is a cause of ciguatera, which is a widespread fish poisoning that presents a variety of neurological symptoms, including hyperalgesia and allodynia
Culture—The wild-type rat Nav1.4 and rat Nav1.2 cDNAs were transiently transfected into HEK293 ity to ciguatoxins of Nav1.8 was derived of the N-terminal half of the Nav1.8 molecule
Ciguatoxin-induced Background Currents through Naϩ Channels—It has been demonstrated that at least 1 ␮M CTX3C is necessary to modulate Nav1.2, Nav1.4, and Nav1.5 channels [21] when the holding potential is kept at Ϫ140 mV

Summary

Introduction

Ciguatoxin is a lipophilic cyclic polyether with 13 ether rings (Fig. 1) derived from the dinoflagellate Gambierdiscus toxicus [1] and is a cause of ciguatera, which is a widespread fish poisoning that presents a variety of neurological symptoms, including hyperalgesia and allodynia. We tested the effects of synthetic ciguatoxin CTX3C on heterologously expressed Nav1.2, Nav1.4, and Nav1.8 channels in either HEK293 or ND7-23 cells. Ciguatoxin-induced Background Currents through Naϩ Channels—It has been demonstrated that at least 1 ␮M CTX3C is necessary to modulate Nav1.2, Nav1.4, and Nav1.5 channels [21] when the holding potential is kept at Ϫ140 mV.

Results

Conclusion