Kv1.2 is a member of the Shaker family of voltage-sensitive potassium channels and contributes to regulation of membrane excitability. The electrophysiological activity of Kv1.2 undergoes tyrosine kinase-dependent suppression in a process involving RhoA. We report that RhoA elicits suppression of Kv1.2 ionic current by modulating channel endocytosis. This occurs through two distinct pathways, one clathrin-dependent and the other cholesterol-dependent. Activation of RhoA downstream effectors Rho-kinase (ROCK) or protein kinase N (PKN) via the lysophosphatidic acid (LPA) receptor elicits clathrin-dependent Kv1.2 endocytosis and consequent attenuation of its ionic current. LPA-induced channel endocytosis is blocked by ROCK inhibition , dominant negative PKN, or by clathrin RNAi. In contrast, steady-state endocytosis of Kv1.2 in un-stimulated cells is cholesterol-dependent. Inhibition of basal ROCK with Y27632 or basal PKN with HA1077 increases steady-state surface Kv1.2. The Y27632-induced increase persists in the presence of clathrin RNAi and, in the presence of the sterol-binding agent filipin, does not elicit an additive effect. Temperature block experiments in conjunction with studies that perturb trafficking of newly synthesized proteins from the Golgi demonstrate that basal ROCK affects cholesterol-dependent trafficking by modulating the recycling of constitutively endocytosed Kv1.2 back to the plasma membrane. Both receptor-stimulated and steady-state Kv1.2 trafficking modulated by RhoA/ROCK require the activation of dynamin as well as the ROCK effector LIM kinase, indicating a key role for actin remodeling in RhoA-dependent Kv1.2 regulation.
| Identifer | oai:union.ndltd.org:uvm.edu/oai:scholarworks.uvm.edu:graddis-1222 |
| Date | 13 February 2009 |
| Creators | Stirling, Lee |
| Publisher | ScholarWorks @ UVM |
| Source Sets | University of Vermont |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate College Dissertations and Theses |
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