In the last 8 years since their discovery, voltage-sensitive phosphatases (VSPs) have represented a fascinating new mechanism of electrochemical coupling in cells. These remarkable proteins contain a voltage sensor domain (VSD) comprised of 4 transmembrane segments, with high sequence homology to those VSDs found in voltage-sensitive ion channels [1]. While activation of this VSD in ion channels results in conformational changes which open the conducting pore of the channel, in VSPs the activation of the VSD results in an increased phosphatase activity towards a phosphoinositide substrate. At present, the best-studied VSPs are those from zebrafish (Dr-VSP) [2] and sea squirt (Ci-VSP) [1]. VSPs from these two organisms have been well-characterized biophysically, and have been used with great effect to probe the influence of phosphoinositides on cellular processes such as ion channel function [3-5]. However, the question of the normal, physiological importance of VSPs, especially in higher mammals such as mice and humans, is as of yet almost completely unexplored. The importance of regulated electrical signaling to a large number of disease states, especially neurological disorders [6], and the known role of phosphoinositide phosphatases in neurological disease pathways [7], indicates the medical importance of fully understanding the role of a protein which couples electrical signaling to phosphoinositide signaling. To address this gap in knowledge, we propose the following three year program of research to elucidate the physiological importance of VSPs, especially with regard to neural signaling. We have already demonstrated, using RT-PCR methods, that the mRNA transcript for the mouse orthologue of Ci-VSP and Dr-VSP (named mTpte, for mouse transmembrane phosphatase with tensin homology) is expressed in the mouse brain, indicating a role of the protein in regulating neural signaling. To further characterize expression in the brain of mice, we will generate an antibody against mTpte for use in immunohistological experiments to determine in which neural cell subtypes the protein is expressed. To characterize the functional capabilities of the protein, we will heterologously express mTpte, and use combined live-cell imaging and electrophysiology to track the depletion of phosphoinositides in response to mTpte activation, as reported by fluorescently-tagged PH domain probes. Finally, we will both knock down expression of endogenous mTpte in neurons using shRNA, and overexpress exogenous mTpte, so that we may perform electrophysiological experiments to characterize the functional effects of mTpte on neural signaling. PUBLIC HEALTH RELEVANCE: We seek to understand the physiological importance of voltage-sensitive phosphatase signaling in neurons, especially as it relates to neurological disease states such as epilepsy. To address this, we are combining a variety of molecular biology, imaging, and biophysics techniques to characterize the expression, regulation, and functionality of voltage-sensitive phosphatases.