Investigating the Function of a Novel Voltage-Sensing Protein

Abstract

We have identified a protein coded by the C15orf27 gene that we named NVS (Novel Voltage Sensor). NVS contains 531 residues, and contains an S1-S4 domain, a 90 residue N-terminus and a 307 residue C-terminus, both of which are predicted to be intracellular. The most critical residues found in S1-S4 domains of other voltage sensors are conserved in NVS, including 3 Arg and a Lys in the S4 helix, 4 conserved acidic residues in S1-S3 and the charge-transfer Phe in S2. In addition, the C-terminus is predicted to contain a coiled-coil domain, similar to voltage-activated proton (Hv1) channels. Our hypothesis is that NVS functions as a voltage sensor that couples to intracellular signaling pathways or interacts with Hv1 to form heteroligomers through the C-terminal coiled-coil domain. We used site-specific voltage-clamp fluorometry and identified several positions at the outer ends of S3 and S4 where labeled Cys residues produced changes in fluorescence as a function of membrane potential. Several positions give complex fluorescence responses, starting with a rapid increase in fluorescence followed by slower decrease in fluorescence. We also investigated whether NVS can oligomerize with Hv1, but observe no change in the gating properties of Hv1 when coexpressed with NVS, and NVS was not capable of interacting with Hv1 in pull down assays. Having no apparent interaction with Hv1, we set out to determine whether NVS forms oligomers. Using chemical crosslinking and pull-down assays, we see formation of oligomers that are consistent with dimers and dependent on the C-terminus. Taken together, our results support the hypothesis that NVS is a voltage sensing protein capable of dimerization, with a function independent of Hv1. We are currently using these findings and approaches to investigate the structural assembly of NVS and to identify interaction partners.