Internalization of the Kv1.4 potassium channel is suppressed by clusteringinteractions with PSD-95

The contribution of voltage-dependent ion channels to nerve function depend s upon their cell-surface distributions. Nevertheless, the mechanisms under lying channel localization are poorly understood. Two phenomena appear part icularly important: the clustering of channels by membrane-associated guany late kinases (MAGUKs), such as PSD-95, and the regional stabilization of ce ll-surface proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined the effect of P SD-95 on the internalization rate of Kv1.4 K+ channels in transfected HEK29 3 cells using cell-surface biotinylation assays. When expressed alone Kv1.4 was internalized with a half-life of 87 min, but, in the presence of PSD-9 5, Kv1.4 internalization was completely suppressed. Immunochemistry and ele ctrophysiology showed PSD-95 had little effect on total or cell-surface lev els of Kv1.4 or on current amplitude, activation, or inactivation kinetics. Clustering was necessary and sufficient to suppress Kv1.4 internalization since C35S-PSD-95, a mutant reported to bind but not cluster Kv1.4, (confir med by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4) restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t(1 /2) = 16 min), These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role for PSD-95, and perha ps other MAGUKs, orchestrating the stabilization of channels at the cell-su rface.