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.
Highlights
Changes in membrane potential that are maintained throughout the duration of a light stimulus [1, 2]
Using FRET experiments, we found that the ICDI binds to the EF-hand motif and downstream sequence of the proximal C terminus
Our finding that the detection of FRET between CaM and Cav1.4 depends on steric orientation would be in favor of slightly different structure of the channel-CaM complex as compared with Cav1.2
Summary
Constructs for Electrophysiology—For expression of murine Cav1.4 [17] (accession number AJ579852), the bicistronic pIRES2-EGFP expression vector (Clontech) was used. Measurements of single-cell FRET based on aggregate (nonspatial) fluorescence recordings were performed using three-cube FRET as described previously [24, 27]. Fluorescence measurements for the determination of SFRET, SCFP, and SYFP were performed in cells coexpressing CFP-tagged and YFP-. Japan) and a built-in dual-emission system (iMIC 2010 FRET stants from measurements applied to single cells expressing module; TILL Photonics). For the single channels (CFP, YFP), mean 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM Na-HEPES, intensity values derived from a selective background region pH 7,4 at room temperature. The ratio (R) of YFP and CFP fluorescence intensities was calculated as the following, R ϭ SFRET/SCFP. For the single channels (CFP, YFP), mean intensity values derived from a selective background region near the investigated cell was used for background correction.
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