Characterization of a Novel Voltage-Sensing Protein

Abstract

The discovery of Ci-VSP, a voltage-sensing phosphatase, revealed that S1-S4 domains can exist in proteins independent of an ion-conducting pore (Nature, 2005). The voltage-activated proton channel, Hv1, was subsequently discovered and shown to consist of a voltage-sensing domain that conducts protons in response to membrane depolarization (Nature, 2006). Through bioinformatic searches, we identified a protein 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 other S1-S4 domains are conserved in NVS, including 3 Arg and a Lys in the S4 helix, and 4 conserved acidic residues in S1-S3. Other than an S1-S4 domain, NVS contains no conserved domains that offer clues about its function. However, the C-terminus does contain a coiled-coil domain, several SH3 binding motifs and a region that has 30% identity with a Ras-GAP binding protein thought to regulate Ras signaling pathways. Here, we show that NVS traffics to the membrane as determined by surface biotinylation. Additionally, tissue distribution studies show expression of NVS in brain, heart, kidney, liver and testes. Within the brain, NVS is enriched in cerebellum and immunofluorescence studies on frozen tissue sections indicate that NVS localizes to pre-synaptic terminals of granule cells. Furthermore, the S4 helix of NVS is capable of sensing changes in membrane potential as revealed by transferring this region into Hv1. Our guiding hypothesis is that NVS functions as a voltage sensor that interacts with signaling proteins to provide intracellular pathways with information about voltage changes across the membrane. To this end, experiments are underway to identify interacting proteins for clues about the function of NVS.