Life science
Medical & healthcare, Biophysics

Molecular mechanisms of electrochemical conversion
in voltage-sensing phosphatase (VSP)
Integrative Physiology, Graduate School of Medicine
Specially Appointed Assistant Professor Natsuki Mizutani

https://researchmap.jp/zdl134skhf?lang=en

Professor Yasushi Okamura						https://researchmap.jp/yasushiokamura?lang=en

Life science

Abstract
Conserved among all organisms, cells utilize plasma membranepotential changes for homeostasis. In neuronal cells, voltage-gated ion channels (VGICs) sense an electrical signal by a voltage
sensor domain (VSD) and translate it into an ionic flow throughthe plasma membrane. Interestingly, in sperm and enterocytes,
voltage-sensing phosphatase (VSP) converts an electrical signal
into phosphoinositide phosphatase activity, which is important for
regulation of sperm motility and nutrient absorption, respectively.
Despite its physiological importance, it remains unclear how anelectrical signal is converted into a chemical one. In this study, we
found that the interaction of the fourth transmembrane segment of
the VSD (S4) with a hydrophobic part of the phosphatase region
called the hydrophobic spine mediates the conversion from anelectrical signal to a chemical one in VSP.

▍ Background & Results
VGIC consists of the VSD and an ion permeation pore. VSP has aVSD similar to that of VGICs but does not have a pore. Instead, theVSD regulates the cytoplasmic phosphatase region with structuralsimilarity to PTEN, a tumor suppressor enzyme, exhibiting the voltage-dependent phosphatase activity toward mainly PI(4,5)P2. We
have reported that the hydrophobic spine plays a critical role in theelectro-chemical signal conversion. However, the detailed mechanism of the conversion remains unclear. To address this issue, wefocused on the C-terminal end of S4 because at that part hydrophobic amino acids (isoleucine and phenylalanine) are conserved
across animal species. We found that mutations of the hydrophobic
part into hydrophilic amino acids diminished the conversion. By
using Ciona intestinalis VSP (Ci-VSP), when we analyzed structural
rearrangements of I233 and F234 replaced with a kind of fluorescent unnatural amino acid, Anap, using voltage clamp fluorometry(VCF) method on Xenopus oocytes, attenuation of I233Anap andF234Anap fluorescence upon membrane depolarization was moreremarkable when tryptophan was introduced in the hydrophobicspine, consistent with the fluorescence quenching. When the hydrophobic spine and the C-terminal end of S4 were replaced bycysteines, restriction of the voltage-dependent S4 motion was induced upon repeated membrane depolarization, indicating that thedisulfide bond was formed between two parts. These are consistent with the idea that voltage-induced upward motion of S4 brings
its C-terminus into close proximity of the hydrophobic spine. The
predicted full-length Ci-VSP structure also supports this interaction.
Taken together, voltage-induced interactions of S4 with the hydrophobic spine play critical roles in translation from an electrical signal into a chemical one in VSP.

▍ Significance of the research and Future perspective
Our findings present a new mechanism underlying information
flow from transmembrane domain to cytoplasmic domain throughhelix to helix interaction. It will help to understand the mechanismsof voltage-gated ion channels of which pathological alteration leads
to various diseases such as arrhythmia and epilepsy. In addition,findings of this work will be of significance for understanding of theregulatory mechanism of PTEN, an important tumor suppressor.

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P a t e n t Japanese Patent No. 4802331
Mizutani, Natsuki; Kawanabe, Akira; Jinno, Yuka et al. Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltageTr e a t i s esensing phosphatase (VSP). Proc Natl Acad Sci U S A (PNAS). 2022, 119 (26), e2200364119. doi: 10.1073/pnas.2200364119
URL https://www.med.osaka-u.ac.jp/pub/phys2/okamura/indexen.html
K e y w o r d ion channel, membrane potential, electrophysiology, electrochemical coupling, unnatural amino acid