Abstract
We previously reported an ascidian protein Ci-VSP which has a transmembrane voltage sensor motif and a phosphatase domain. We showed that the voltage sensor domain functionally couples with the phosphatase domain and the phosphatase activity arises by depolarization. We also showed that the linker region between the voltage sensor domain and phosphatase domain plays an important role in this coupling. However, these residues found in Ci-VSP are not completely conserved in zebrafish VSP (z-VSP) that also shows activities of voltage-regulated phosphatase. We studied structure-function relationship of the coupling.First, to test if the coupling mechanism is common to other species, residues in the linker of Ci-VSP that are distinct from those of z-VSP were changed into z-VSP-based ones. Next, we tested whether the length of the linker affects coupling by introducing additional residues or deleting some residues around the linker. If the length of the linker is important, these mutants may not express the voltage-dependent change. Moreover, we tested whether there is interaction between the linker and the enzymatic domain by swapping the linker and the proximal enzymatic domain between Ciona and zebrafish VSPs. Wild-type and mutant VSPs were coexpressed with phosphoinositide-sensitive GIRK2 channels into Xenopus oocytes, and changes of the phosphatase activity with membrane potentials were examined by measuring GIRK2 channel activity using the two-electrode voltage clamp recording. [J Physiol Sci. 2007;57 Suppl:S228]
