Internalization of the Kv1.4 Potassium Channel Is Suppressed by Clustering Interactions with PSD-95*
- From the ‡Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada, the §Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and the¶Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Abstract
The contribution of voltage-dependent ion channels to nerve function depends upon their cell-surface distributions. Nevertheless,
the mechanisms underlying channel localization are poorly understood. Two phenomena appear particularly important: the clustering
of channels by membrane-associated guanylate kinases (MAGUKs), such as PSD-95, and the regional stabilization of cell-surface
proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined
the effect of PSD-95 on the internalization rate of Kv1.4 K+ channels in transfected HEK293 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-95, Kv1.4 internalization was completely suppressed. Immunochemistry
and electrophysiology showed PSD-95 had little effect on total or cell-surface levels 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, (confirmed by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4)
restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t
= 16 min). These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role
for PSD-95, and perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.
Footnotes
-
↵* This work was supported by awards from the Natural Sciences and Engineering Research Council of Canada (to O. T. J), the Medical Research Council of Canada (to L. C. S), a studentship from the Bloorview Epilepsy Program (to D. G. M. J.), and a Santalo scholarship from the University of Toronto (to R. K).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‖ To whom correspondence should be addressed: Div. of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, MC 11-434, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.: 416-603-5039; Fax: 416-603-5745; E-mail: owen@playfair.utoronto.ca.
- Abbreviations:
- MAGUK
-
membrane-associated guanylate kinase
- PSD-95
-
postsynaptic density-95
- PCR
-
polymerase chain reaction
- EYFP
-
extended yellow variant of green fluorescent protein
- HEK293
-
human embryonic kidney cells
- PBS-CM
-
phosphate-buffered saline
- ECL
-
enhanced chemiluminescence
- BAPTA
-
1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
-
- Received July 12, 1999.
- Revision received October 7, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
