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, and that the proximal 8 amino acid in the linker region between the voltage sensor domain and phosphatase domain plays an important role in this coupling. In this report, we further examined this coupling by functional expression of Ci-VSP in Xenopus oocyte. First, we deleted the most proximal or distal half of the 8 amino acid linker segment and also made some point mutants. cRNAs encoding these mutants were coinjected with GIRK2 phosphoinositide-sensitive ion channels into Xenopus oocyte and the two-electrode voltage clamp recording was performed. Changes of the phosphatase activity with membrane potentials were detected by monitoring changes of ion currents through GIRK2 channels. Some mutants showed no significant change of the amplitude of GIRK currents. Secondly, we analyzed the voltage dependency of the coupling with mutant constructs in which properties of voltage sensor movement are altered. Some mutants showed shift of the Q-V curve of gating current. We are currently testing whether voltage-dependency of phosphatase activity is similarly shifted in those mutants. [Jpn J Physiol 55 Suppl:S124 (2005)]
