Dynamic structural rearrangements and functional regulation of voltage-sensing phosphatase.

The voltage-sensing phosphatase, VSP, consists of the voltage sensor domain (VSD) and the cytoplasmic catalytic region. The latter contains the phosphatase domain and the C2 domain, showing remarkable similarity to a tumor suppressor enzyme, PTEN. In VSP, membrane depolarization induces the conformational change in the VSD, which activates the phosphoinositide phosphatase. The final outcome of VSP is enzymatic activity of the cytoplasmic region unlike voltage-gated ion channels where conformational change of the transmembrane pore is induced by the VSD. Therefore, it is crucial to detect structural change of the cytoplasmic catalytic region to gain insights into operating mechanisms of VSP. This review summarizes a recent study of applying a method of genetic incorporation of a noncanonical amino acid, Anap, to detect dynamic rearrangements of the structure of the catalytic region of sea squirt VSP (Ci-VSP) under control of membrane voltage. Both the phosphatase domain and the C2 domain move in a similar timing upon membrane depolarization, suggesting that the two regions are coupled to each other. Measurement of FRET between Anap introduced into the C2 domain of Ci-VSP and dipicrylamine in cell membrane suggested no large motion of the enzyme toward the membrane. Fluorescence change of Anap induced by different membrane potentials indicates the presence of multiple conformations of the active enzyme. A fluorescent unnatural amino acid, Anap, was genetically incorporated into the catalytic domain of Ci-VSP in Xenopus oocytes. The fluorescence uncovered the conformation change associated with catalytic activity of Ci-VSP. This article is protected by copyright. All rights reserved.