Voltage sensing phosphatase, VSP, contains four transmembrane helices that operates as a voltage sensor (VS) and a cytoplasmic catalytic region (CCR) with remarkable similarity to PTEN, a tumor suppressor phosphatase1. VSP is highly conserved from marine invertebrates to human, playing roles in the regulation of sperm maturation and epithelial functions. In contrast to voltage-gated ion channels where the VS regulates the transmembrane pore-gated domain, the VS of VSP activates the PI (4,5) P2 phosphatase, which is the C-terminal cytoplasmic region. We previously reported a hydrophobic structure within the CCR of Ciona intestinalis VSP (Ci-VSP) that protrudes into the membrane, and call this structure the “hydrophobic spine (HS).” The HS plays key roles in both the enzyme activity and coupling to the VS2. However, little is known on how the VS regulates the HS in VSP so far.
In this study, we found by mutagenesis screening that the hydrophobicity of two residues, I233 and F234, positioned just below the lowest arginine residue of S4, is critical for the coupling. Detailed motions of I233, F234 and the neighboring residues were studied using the method of incorporating Anap, a fluorescent unnatural amino acid. Voltage-induced fluorescence signals derived from Anap introduced at these sites were remarkably reduced when tryptophan was introduced at the HS. Furthermore, upon repeated membrane depolarizations, mutants with double cysteines on the S4 and HS showed remarkable reduction in sensing currents derived from the motion of the VS. This result provides evidence that the two regions come closer to each other with the upward motion of the VS. We propose a model of coupling between the VS and CCR where the lower hydrophobic part of the S4 forms a hydrophobic core with the HS at the membrane interface, activating the C-terminal enzyme region. Given that HS-like structure is conserved among many phosphoinositide phosphatases including PTEN, which does not contain a VS, moving the VS of VSP may perhaps “hijack” regulatory mechanisms innate to PTEN-like phosphoinositide phosphatases.
 

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