Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltage-sensing phosphatase (VSP)

Abstract

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP.

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

“…Anap has been incorporated in the S4 of the hyperpolarization-activated cyclic nucleotide-gated channel ( Dai et al, 2019 ), where it is quenched or dequenched upon hyperpolarization in a position-dependent manner. It has also recently been incorporated at the bottom of the S4 in the voltage-dependent phosphatase, CiVSP ( Mizutani et al, 2022 ), where it becomes quenched upon depolarization. The direction of the fluorescence changes due to S4 motion is difficult to predict since, as we have shown, Anap’s fluorescence can be affected by both the local environment’s polarity and interaction with specific quenching groups that are part of the channel sequence.…”

Structural change of the cytoplasmic N‐terminus and S1 segment of voltage‐sensing phosphatase reported by Anap

“…As a trend in S1, the residues at the intracellular side tended to exhibit fluorescence increase except for L125 and those at the extracellular side exhibited fluorescence decrease (Figure 2, side view). This trend is not applied to S4 since A231Anap in this study as well as I233Anap and F234Anap in the previous report 11 generated a fluorescence decrease upon the depolarization. There was no clear correlation between the polarity of the signal and the orientation of the residues in the crystal structure.…”

“…To do site‐specific labeling of VSD with Anap or TMR, the position of interest was replaced by amber or cysteine codon, respectively. The protocol for site‐directed mutagenesis has been described elsewhere 11 …”

“…On the other hand, the VSD isolated from VSP is functionally independent, exhibiting robust gating currents in the heterologous expressions. VSP, therefore, serves as a model for studying the nature of VSD as well as VSD-effector coupling, 3,[9][10][11][12][13] and is also significant in developing molecular tools. 14 While various approaches have been taken to understand the nature of VSP and its VSD as well, their biophysical pictures have not been fully resolved.…”

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

“…Anap has been incorporated in the S4 of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel (Dai et al, 2019), where it is quenched or dequenched upon hyperpolarization in a position-dependent manner. It has also recently been incorporated at the bottom of the S4 in the voltage-dependent phosphatase, CiVSP (Mizutani et al, 2022), where it becomes quenched upon depolarization. The direction of the fluorescence changes due to S4 motion are difficult to predict, since, as we have shown, Anap’s fluorescence can be affected by both the local environment’s polarity and interaction with specific quenching groups that are part of the channel sequence.…”