Exploring Conformational Rearrangements in a Novel Voltage-Sensing Protein

Abstract

Through bioinformatic searches, we have identified a protein coded by the C15orf27 gene that we named NVS (Novel Voltage Sensor). NVS contains 531 residues and consists of 3 parts: 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 Hv1. Our working hypothesis is that NVS functions as a voltage sensor that couples to intracellular signaling pathways (as yet undefined) or interacts with Hv1 to form hetero-oligomers through the C-terminal coiled-coil domain. In the present study we used site-specific voltage-clamp fluorometry to look for evidence that NVS may undergo conformational rearrangements in response to changes in membrane potential. We identified several positions on the S3-S4 loop where introduced and 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. Additionally we applied fluorescence quenchers extracellularly and examined the voltage dependence of quenching. Our results show that depolarization increases fluorescence quenching, suggesting the quencher has increased access to the fluorophore at positive membrane potentials. Taken together, our results support the hypothesis that NVS undergoes a conformational change in responses to membrane depolarization, and we are currently investigating the oligomeric state of NVS testing whether it can form heteromeric complexes with Hv1.