Abstract
Voltage-gated ion channels are composed of six transmembrane segments (S1 to S6). S5 and S6 form the hydrophilic pore, while S1 to S4 constitute the voltage-sensor domain (VSD). We have recently identified a novel protein that consists only of the four-transmembrane segments, corresponding to VSD, and found that it is long-sought molecular correlate for voltage-gated proton channel (VSOP) (Sasaki et al, Science 2006). Voltage-gated potassium channels are known to assemble as tetramers. However, it remains unknown whether VSOP functions as multimers or monomers. In this study, we have investigated the protein stoichiometry of VSOP labeled with green fluorescent protein (GFP-VSOP) by total internal reflection fluorescence microscopy. GFP-VSOP was overexpressed in Xenopus oocyte and fluorescence intensity and whole-cell current were simultaneously measured under the two-electrode voltage clamp. This protein showed depolarization-induced outward currents and voltage-dependent fluorescence change probably due to local change of pH. In this chimeric protein, activation kinetics was faster and deactivation was slower than in the wild-type protein. This suggests that fusion of GFP to VSOP slightly modifies properties of channel activities. We are currently using this molecule to identify the stoichiometry of GFP-VSOP by the photobleaching of single GFP molecules. [J Physiol Sci. 2007;57 Suppl:S228]
