Is the Skeletal Muscle Sodium Channel of the Batrachotoxin (BTX)-Producing Phyllobates aurotaenia Poison Dart Frog Resistant to BTX?

Abstract

Batrachotoxin is a potent toxin found in skins of Phyllobates frogs. The skeletal muscle Na+ channels of Phyllobates aurotaenia frogs have been proposed to be resistant to high concentrations of BTX (>1μM). In order to unravel the mechanism and structural elements that confer BTX-resistance to P. aurotaenia, we cloned its skeletal muscle Na+ channel, PaNaV1.4. As reported last year, PaNaV1.4 has high homology (>70%) with other NaV1.4 channels especially in the membrane spanning regions. Some residues that have been identified in mutagenesis studies as critical for BTX-channel interaction in mammalian NaV are conserved in PaNaV1.4. To further address the issue, we have expressed PaNaV1.4 in Xenopus laevis oocytes. We report here the functional characterization of PaNaV1.4, studied under voltage-clamp, and its response to BTX.PaNaV1.4 expresses robustly. While the general characteristics of the ionic currents were similar, at room temperature PaNaV1.4 tended to open at more depolarized voltages, inactivated faster and currents peaked earlier than rNaV1.4. BTX, at concentrations as high as 10μM, had a significantly lower effect on PaNaV1.4 currents than on rNaV1.4. The ratio of plateau to peak currents at 80 mV was ∼0.2-0.5 in PaNaV1.4 while >0.95 in rNaV1.4. BTX modification of PaNaV1.4 occurred at a slow rate. Both activation thresholds were negatively shifted. Because most of the residues proposed to participate in the BTX effect are located in the pore lining segment (S6) of NaV, we have also studied Pa/rNaV1.4 hybrid channels with domains, S6 segments or residues swapped or exchanged.Supported by COLCIENCIAS1106-12-13836 (LF), AHA 0725763Z (WS), and NIH GM030376 (FB) and GM068044 (AMC).