Phosphorylation of the Kv2.1 K+ Channel Alters Voltage-Dependent Activation
- Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215
Abstract
The voltage-gated delayed-rectifier-type K+ channel Kv2.1 is expressed in high-density clusters on the soma and proximal dendrites of mammalian central neurons; thus, dynamic regulation of Kv2.1 would be predicted to have an impact on dendritic excitability. Rat brain Kv2.1 polypeptides are phosphorylated extensively, leading to a dramatically increased molecular mass on sodium dodecyl sulfate gels. Phosphoamino acid analysis of Kv2.1 expressed in transfected cells and labeled in vivo with 32P shows that phosphorylation was restricted to serine residues and that a truncation mutant, ΔC318, which lacks the last 318 amino acids in the cytoplasmic carboxyl terminus, was phosphorylated to a much lesser degree than was wild-type Kv2.1. Whole-cell patch-clamp studies showed that the voltage-dependence of activation of ΔC318 was shifted to more negative membrane potentials than Kv2.1 without differences in macroscopic kinetics; however, the differences in the voltage-dependence of activation between Kv2.1 and ΔC318 were eliminated by in vivo intracellular application of alkaline phosphatase, suggesting that these differences were due to differential phosphorylation. Similar analyses of other truncation and point mutants indicated that the phosphorylation sites responsible for the observed differences in voltage-dependent activation lie between amino acids 667 and 853 near the distal end of the Kv2.1 carboxyl terminus. Together, these parallel biochemical and electrophysiological results provide direct evidence that the voltage-dependent activation of the delayed-rectifier K+ channel Kv2.1 can be modulated by direct phosphorylation of the channel protein; such modulation of Kv2.1 could dynamically regulate dendritic excitability.
Footnotes
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Send reprint requests to: Dr. James S. Trimmer, Department of Biochemistry and Cell Biology, SUNY at Stony Brook, Stony Brook, NY 11794-5215. E-mail:trimmer{at}life.bio.sunysb.edu
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↵1 Current affiliation: Hormone Research Institute, University of California, San Francisco, CA 94143.
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↵2 K. L. Lopez and J. S. Trimmer, unpublished observations.
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This work was supported by a Grant-in-Aid from the American Heart Association, New York State Affiliate, Inc. (H.M.), and by National Institutes of Health Grants NS34375 and NS34383 (J.S.T.). This work was done during the tenure of an Established Investigatorship from the American Heart Association (J.S.T.).
- Abbreviations:
- AP
- alkaline phosphatase
- DMEM
- Dulbecco’s modified Eagle’s medium
- PKA
- cAMP-dependent protein kinase
- CNBr
- cyanogen bromide
- MDCK
- Madin-Darby canine kidney
- V½
- membrane potential for half-activation potential
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide electrophoresis gel
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received June 20, 1997.
- Accepted July 22, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
