Enzyme Domain Affects the Movement of the Voltage Sensor in Ascidian and Zebrafish Voltage-sensing Phosphatases

Abstract

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN).The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON-and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.Voltage-gated ion channels play an important role in electrical activities and cell signaling of muscles and nerves. The first four transmembrane regions (S1-S4) are conserved among all the voltage-gated channels and operate as the voltage sensor, thus called the voltage sensor domain (VSD) 4 (1). The VSD regulates the operation of the downstream pore domain, consisting of the two transmembrane segments (S5-S6) and a loop that provides an ion permeation pathway. The VSD has several positively charged residues interspersed with two hydrophobic residues in the fourth transmembrane segment, called S4, that plays critical roles in voltage sensing (1-4). Recently resolution of the crystal structure of voltage-gated potassium channels (5, 6) and biophysical measurements of the movement of specific sites of the VSD (3) have led to proposed models for the operation of the VSD (7). However, the VSD has long been studied as a structure unique to voltage-gated ion channels.We have recently identified a protein, Ci-VSP, that contains the VSD but not the pore domain (8). Ci-VSP has the following two modules in its structure: the transmembrane spanning region that corresponds to the VSD of voltage-gated ion channels and the cytoplasmic region with homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN), a PtdIns(3,4,5)P 3 phosphatase (9). The VSD of Ci-VSP exhibits gating currents that indicate the conformational change in response to membrane voltage, and the phosphoinositide phosphatase activity is voltage-dependently regulated (8). This is the first ...

Dynamic PIP 2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

“…1A). Next, we decreased the PIP 2 concentration using a voltage-sensitive phosphatase from Danio rerio (Dr-VSP), which hydrolyzes PIP 2 at highly depolarized voltages (e.g., +120 mV) and transiently reduces the PIP 2 level (28). In the cells cotransfected with KCNQ2 and Dr-VSP, the current is significantly reduced upon +120 mV depolarization (Fig.…”

A voltage-sensing phosphatase, Ci-VSP, which shares sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5-bisphosphate

“…However, Gly-365 in Ci-VSP is conserved in all mammalian VSPs, suggesting that mammalian VSPs may also dephosphorylate PI(4,5)P 2 . A recent study indi- cated that PI(4,5)P 2 is dephosphorylated by the teleost VSP (25). Further studies will be necessary to address whether substrate specificity of Ci-VSP is conserved in mammals.…”

A human phospholipid phosphatase activated by a transmembrane control module

“…Further differences in the catalytic center may also contribute to the distinct enzymatic specifi cities; this remains to be addressed. Despite this complexity, all CX 5 R-type 5-phosphatases examined to date display the critical G variant motif ( 2,8,9,26 ), suggesting that not only hVSP1, but also yet-uncharacterized VSP homologs from other mammalian species, are 5-phosphatases, because all share the G variant.…”