Impact of S1 hydrophobic residues on voltage sensing

Abstract

The voltage sensing domain (VSD) is a modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a unique protein that dephosphorylates phosphatidylinositol phosphates (PIPs) in a voltage dependent manner. VSP dimers were recently identified where both the VSD and phosphatase domain (PD) contributed to dimerization. The tight coupling between the VSD and PD in VSP is well established and the contribution of S1-S3 to enzymatic activity has not been explored. We investigated whether the S1 in VSP contributed to function by mutating several amino acids in S1 based on the solved Ci-VSD structure interface. Using co-immunoprecipitation, we found that none of the mutations broke the dimer. The same mutations consistently shifted the voltage dependence of activity to higher voltages. The kinetics of enzymatic activity were also altered with some cases significantly slowing down dephosphorylation. The voltage dependence of VSD motions were altered with these mutations, shifting them to lower voltages and revealing a second motion. Lastly, when the alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme and influencing the function of the VSD.