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Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.

ABSTRACT: Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the selectivity of the actions of CaMKII among Ca2+ channel beta subunit isoforms. CaMKII phosphorylates the beta1b, beta2a, beta3, and beta4 isoforms with similar initial rates and final stoichiometries of 6-12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to beta1b and beta2a with a similar apparent affinity but does not bind to beta3 or beta4. Prephosphorylation of beta1b and beta2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in beta2a are highly conserved in beta1b but are different in beta3 and beta4. Site-directed mutagenesis of this domain in beta2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-beta2a complexes in vitro and reduces interactions of CaMKII with beta2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with beta2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca2+ channel beta subunits with CaMKII.

SUBMITTER: Grueter CE

PROVIDER: S-EPMC2814322 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.

Grueter Chad E CE Abiria Sunday A SA Wu Yunji Y Anderson Mark E ME Colbran Roger J RJ

Biochemistry 20080119 6

Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the se ...[more]

PMID: 18205403

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Project description:As with other elements of the peripheral auditory system, spiral ganglion neurons display specializations that vary as a function of location along the tonotopic axis. Previous work has shown that voltage-gated K(+) channels and synaptic proteins show graded changes in their density that confers rapid responsiveness to neurons in the high frequency, basal region of the cochlea and slower, more maintained responsiveness to neurons in the low frequency, apical region of the cochlea. In order to understand how voltage-gated calcium channels (VGCCs) may contribute to these diverse phenotypes, we identified the VGCC α-subunits expressed in the ganglion, investigated aspects of Ca(2+)-dependent neuronal firing patterns, and mapped the intracellular and intercellular distributions of seven VGCC α-subunits in the spiral ganglion in vitro. Initial experiments with qRT-PCR showed that eight of the ten known VGCC α-subunits were expressed in the ganglion and electrophysiological analysis revealed firing patterns that were consistent with the presence of both LVA and HVA Ca(2+) channels. Moreover, we were able to study seven of the α-subunits with immunocytochemistry, and we found that all were present in spiral ganglion neurons, three of which were neuron-specific (Ca(V)1.3, Ca(V)2.2, and Ca(V)3.3). Further characterization of neuron-specific α-subunits showed that Ca(V)1.3 and Ca(V)3.3 were tonotopically-distributed, whereas Ca(V)2.2 was uniformly distributed in apical and basal neurons. Multiple VGCC α-subunits were also immunolocalized to Schwann cells, having distinct intracellular localizations, and, significantly, appearing to distinguish putative compact (Ca(V)2.3, Ca(V)3.1) from loose (Ca(V)1.2) myelin. Electrophysiological evaluation of spiral ganglion neurons in the presence of TEA revealed Ca(2+) plateau potentials with slopes that varied proportionately with the cochlear region from which neurons were isolated. Because afterhyperpolarizations were minimal or absent under these conditions, we hypothesize that differential density and/or kinetics of one or more of the VGCC α-subunits could account for observed tonotopic differences. These experiments have set the stage for defining the clear multiplicity of functional control in neurons and Schwann cells of the spiral ganglion.

Dataset Information

Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.

Publications

Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.

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