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Stable membrane expression of postsynaptic CaV1.2 calcium channel clusters is independent of interactions with AKAP79/150 and PDZ proteins.

ABSTRACT: In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of Ca(V)1.2 and its association with upstream and downstream signaling proteins is important for efficient and specific signal transduction. Here we tested the hypothesis that A-kinase anchoring proteins (AKAPs) or PDZ-proteins are responsible for the targeting and anchoring of Ca(V)1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged Ca(V)1.2 demonstrated that clusters of membrane-incorporated Ca(V)1.2-HA were colocalized with AKAP79/150 but not with PSD-95 in the spines and shafts of dendrites. To disrupt the interactions with these scaffold proteins, we mutated known binding sequences for AKAP79/150 and PDZ proteins in the C terminus of Ca(V)1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant Ca(V)1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca(V)1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and alpha-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of Ca(V)1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca(V)1.2 calcium channels form signaling complexes apart from those of glutamate receptors and PSD-95. Their number and distribution in dendritic spines is not altered upon NMDA-induced disruption of the glutamate receptor signaling complex, and targeting and anchoring of Ca(V)1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.

SUBMITTER: Di Biase V

PROVIDER: S-EPMC2853706 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K(+) transport, and are regulated by numerous receptors via G(q/11)-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, and M current is known to be regulated by Ca(2+)/calmodulin (CaM) and PIP(2), we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca(2+)/CaM and PIP(2) on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M(1) receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to "sensitize" KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose-response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP(2) in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca(2+), to disrupt the interaction of YFP-tagged AKAP79(1-153) with CFP-tagged KCNQ2-5. Tonic depletion of PIP(2) by cotransfection of a PIP(2) phosphatase had no effect, and sudden depletion of PIP(2) did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP(2) depletion.

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Stable membrane expression of postsynaptic CaV1.2 calcium channel clusters is independent of interactions with AKAP79/150 and PDZ proteins.

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