University of Vermont COBRE 1 (P20 RR016435)
"Center for Neuroscience Excellence"
Research Project 3: "Kinase and Cytoskeletal Regulation of Potassium Channels"
Investigator: Anthony Morielli, Ph.D.
Our goal is to describe, at the molecular level, the cellular mechanism of tyrosine phosphorylation dependent regulation of an ion channel. We will focus on the voltage dependent potassium channel Kv1.2 since tyrosine phosphorylation of the alpha subunit of Kv1.2 suppresses its ability to generate ionic current. Our previous research suggests a role for the small GTP-binding protein RhoA, as well as the cytoskeleton in this process. We will therefore test the hypothesis that tyrosine phosphorylation dependent suppression of Kv1.2 occurs by a mechanism that involves the cytoskeleton. To accomplish this, we will take thee complementary approaches. First, we will test the hypothesis that RhoA mediated Kv1.2 suppression occurs via RhoA effects on the actin cytoskeleton. Second, we will test the hypothesis that channel activity is affected by direct channel-cytoskeleton interactions, and that channel tyrosine phosphorylation alters those interactions, resulting in channel suppression. Finally, we will extend our studies from tissue culture model systems to cerebral artery vascular smooth muscle, a native tissue expressing endogenous Kv1.2. This will allow us to test whether the ideas we have developed using cellular model systems extend to a physiologically normal system. It also has the powerful advantage of allowing us to apply our findings regarding Kv1.2 regulation towards the understanding of a clinically important human pathology, namely hemorrhage induced cerebral artery vasospasm. To accomplish these goals we will use a combination of molecular biological, microscopy, biochemical, and electrophysiological techniques to determine the relationships that exist between signal induced Kv1.2 suppression, signal, signal induced effects on the actin cytoskeleton, and signal induced effects on the direct interaction between Kv1.2 and the cytoskeleton. The experiments proposed here will, if successful, establish a detailed molecular model of Kv1.2 suppression, identify a new paradigm for channel modulation (i.e. tyrosine phosphorylation dependent channel interactions with the cytoskeleton), verify that model in a native tissue system, and provide the basis for new avenues of research into the understanding of the clinically important phenomenon of hemorrhage induced cerebral artery vasospasm.