Coupling Mechanisms of VSD Mutants of Ci-VSP

Abstract

Voltage-sensing phosphatase (VSP) shows phosphatase activity toward phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) upon membrane depolarization. Voltage sensor deficient mutants lack phosphatase activity and mutations in a short linker between voltage sensor domain (VSD) and the PTEN-like phosphatase domain reduce phosphatase activity, suggesting that activation of VSD “couples” with phosphatase activity via the linker. However, it is unclear how VSD regulates phosphatase activity. We have recently found that amino acid mutation at F234 (two amino acids downstream of the 4th arginine (R4) of S4) in Ciona intestinalis VSP (Ci-VSP) causes a remarkable decrease in the phosphatase activity whereas the gating current is normal. To understand what structural detail of VSD is optimal for inducting enzyme activity in Ci-VSP, we analyzed both VSD motions and enzyme activities of F234 mutants and other VSD mutants. Mutants of Ci-VSP were expressed in Xenopus oocytes and analyzed by the two-electrode voltage clamp. We studied the voltage-driven motion of VSD or cytoplasmic catalytic region with thiol-reactive fluorescent dye or with unnatural fluorescent amino acid, Anap, respectively. Surprisingly, in multiple mutants including F234 mutant, phosphatase activity remarkably decreased whereas the gating current remained intact. We interpret these findings based on the hypotheses: (1) VSD cannot be fully activated, leading only partial phosphatase activity, (2) the structure of the fully-activated VSD differs from that of the normal full activation state, failing to induce the full phosphatase activity, (3) local membrane structure or environment facing the cytoplasmic side of the VSD may be altered by the mutations, possibly preventing the interaction between enzyme region and the plasma membrane which may be critical for phosphatase activity (the second and the third idea are not exclusive to each other).