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Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation.

ABSTRACT: The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase II? (CaMKII?) in cells. Although the C-terminal domain selectively binds CaMKII? in vitro, full-length densin associates with CaMKII? or CaMKII? in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKII?/? catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.

SUBMITTER: Jiao Y

PROVIDER: S-EPMC3137056 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation.

Jiao Yuxia Y Jalan-Sakrikar Nidhi N Robison A J AJ Baucum Anthony J AJ Bass Martha A MA Colbran Roger J RJ

The Journal of biological chemistry 20110524 28

The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr ...[more]

PMID: 21610080

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Project description:TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159-212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487-517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816-824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015-1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525-3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of approximately 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.

Dataset Information

Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation.

Publications

Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation.

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