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Calmodulin is a functional regulator of Cav1.4 L-type Ca2+ channels.

ABSTRACT: Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects.

SUBMITTER: Griessmeier K

PROVIDER: S-EPMC2785611 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Calmodulin is a functional regulator of Cav1.4 L-type Ca2+ channels.

Griessmeier Kristina K Cuny Hartmut H Rötzer Katrin K Griesbeck Oliver O Harz Hartmann H Biel Martin M Wahl-Schott Christian C

The Journal of biological chemistry 20090828 43

Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of r ...[more]

PMID: 19717559

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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.

Dataset Information

Calmodulin is a functional regulator of Cav1.4 L-type Ca2+ channels.

Publications

Calmodulin is a functional regulator of Cav1.4 L-type Ca2+ channels.

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