Abstract
We previously reported an ascidian protein Ci-VSP which has a transmembrane voltage sensor motif with significant homology to voltage-gated channels and a phosphatase domain just downstream of the transmembrane region. We showed that the voltage sensor functionally couples with the phosphatase domain (Nature, 2005). However, it remains unknown whether similar mechanisms for voltage sensing of voltage-gated channels operate in Ci-VSP.There are conserved negative-charged residues in S2 and S3 regions of both Ci-VSP and voltage-gated ion channels. These residues are known to contribute to the gating charge of the channels. We have previously shown that a mutation which neutralized the negative-charged residue in S2 altered voltage dependency of its gating current. In this mutant, voltage-dependence of the coupling was shifted in the same direction as the change of the Q-V curve. In this work, we systematically mutated negatively charged residues in S1-S3 regions of Ci-VSP. cRNAs encoding these mutants were expressed in Xenopus oocytes and the two-electrode voltage clamp recording was performed to record the gating current of these mutants. In addition, these mutants were co-expressed with GIRK2 channels and changes of the phosphatase activity with membrane potentials were detected by monitoring changes of ion currents through GIRK2 channels. We also analyzed the voltage dependence of phosphatase activity of the mutant constructs of which the properties of voltage sensor movement are altered. [J Physiol Sci. 2006;56 Suppl:S157]
