Internalization of the Kv1.4 potassium channel is suppressed by clustering interactions with PSD-95.
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((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 perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.
Jugloff
D G
DG
Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada.
Khanna
R
R
Schlichter
L C
LC
Jones
O T
OT
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D000076122:Disks Large Homolog 4 Protein; D004705:Endocytosis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D051665:Kv1.4 Potassium Channel; D008564:Membrane Potentials; D008565:Membrane Proteins; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000737:chemistry / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014162:Transfection
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; C496977:KCNA4 protein, human; C496979:Kcna4 protein, rat; D051665:Kv1.4 Potassium Channel; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; C033652:postsynaptic density proteins