JGP study (Rayaprolu et al. https://doi.org/10.1085/jgp.202313467) shows that hydrophobic residues in the S1 transmembrane domain modulate the voltage sensor movements and enzymatic activity of voltage-sensing phosphatase.

Best known for their role in voltage-gated ion channels, voltage-sensing domains (VSDs) are protein modules that regulate protein activity in response to electrical signals. Of the four transmembrane helices that make up the VSD, the fourth helix, S4, is considered to be the most important as it contains charged arginine residues that shift the helix’s position in response to changes in membrane potential. In contrast, the other three helices, S1–S3, are generally thought to form an immobile scaffold that has little effect on voltage sensitivity (1). In this issue of JGP, however, Rayaprolu et al. reveal that, at least in the voltage-sensing phosphatase (VSP), hydrophobic residues in the S1 helix play a key role in modulating not only the voltage-dependent movements of the S4 helix, but also the activity and kinetics of the enzyme’s catalytic domain (2).

Vamseedhar Rayaprolu and Susy Kohout.

VSP is composed of a VSD linked to a phosphatase domain that can remove the 3- and 5-phosphates from the inositol ring of phosphatidylinositol phosphates (PIPs) in a voltage-dependent manner. Previous studies have shown that VSP can form dimers and that this might be mediated, in part, by the S1 helices of each subunit, which face each other at the dimer interface (3, 4). Susy Kohout, an associate professor at Cooper Medical School of Rowan University, therefore wondered whether S1 might play a more significant role in VSP than it does in voltage-gated ion channels, where the VSDs are not involved in intersubunit interactions. “It doesn’t make sense that the S1 of VSP isn’t doing anything when the crystal structure shows that it’s located in that position,” Kohout says.

To investigate the importance of S1 in VSP, Kohout and colleagues, including first author Vamseedhar Rayaprolu, selected four hydrophobic residues that line the subunit interface, and mutated them, individually or in combination, to alanine (2). The researchers then expressed the wild-type and mutant phosphatases in Xenopus oocytes and analyzed how the mutations affected the magnitude, kinetics, and voltage dependence of enzyme activity, monitoring the various lipid substrates and products of VSP with specific biosensors.

Three of the four individual mutants had significant, albeit small, effects on VSP function, slowing the kinetics of some dephosphorylation reactions and shifting the voltage dependence of activity to higher voltages. The other individual mutation, of a highly conserved leucine residue in S1, had a more dramatic effect, reducing the magnitude and kinetics of every VSP-catalyzed reaction, as well as shifting the activity to higher voltages. Combining all four mutations had a still stronger effect. “This suggests that the entire VSD—including S1—is transmitting information to the catalytic domain. It’s not just the S4 movement,” Kohout says.

The VSD of voltage-sensing phosphatase can dimerize with the S1 (orange) and S4 (green) helices facing each other at the subunit interface. Rayaprolu et al. (2) reveal that hydrophobic resides (blue) in the S1 helix modulate both the voltage-sensitive movement of the S4 helix and the enzyme’s catalytic activity, suggesting that they play a key role in mediating communication between the protein’s voltage-sensing and phosphatase domains.

Indeed, when the researchers used voltage-clamp fluorometry to monitor the movements of S4, they found that mutations in S1 consistently altered the voltage dependence of these motions to lower voltages, even as enzymatic activity was shifted to higher voltages. “Mutations in S1 seem to weaken the communication between the VSD and catalytic domains,” Kohout explains. “S4 can move more easily in these mutants, but it needs to move more to activate the enzyme.”

None of the S1 mutants prevented VSP from dimerizing, so it remains to be seen whether signals from the VSD to the phosphatase domain are transmitted between subunits, within subunits, or both. Addressing this question will require the development of methods to both disrupt and force VSP dimerization. “If we want to understand this protein, we have to be able to separate these possibilities out,” Kohout says.

1.
Islas
,
L.D.
2016
.
Channels
.
2.
Rayaprolu
V.
, et al
.
2024
.
J. Gen. Physiol.
3.
Li
,
Q.
, et al
.
2014
.
Nat. Struct. Mol. Biol.
4.
Rayaprolu
,
V.
, et al
.
2018
.
J. Gen. Physiol.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).