A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.
Bottom Line: Neutralization of four arginine residues eliminated plasma membrane binding.Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane.Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.Show MeSH
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Mentions: We also tested whether the arginine mutations in CaV1.2S4E (versus CaV1.2S as wild-type control) affect the time-dependent decline of whole-cell ICa and the modulation by added PIP2 (by intracellular application of a 100 μm concentration of the water-soluble PIP2 analogue diC8-PIP2, (15, 64)) or phosphoinositide hydrolysis induced by wortmannin (20 μm) plus m-3M3FBS (50 μm) as in live cell imaging experiments (Fig. 5A). Perfusion of cells with control solution induced a slow decrease in activity with time (“run-down”), as expected for Cav1.2 channels (65). ICa decline during perfusion with control solution was similar in wild-type and the mutant channel and was also not affected by intracellular application of diC8-PIP2 (Fig. 7). However, extracellular perfusion with wortmannin/m-3M3FBS significantly inhibited ICa of wild-type and Cav1.24E channels, but this current inhibition was significantly attenuated in the mutant channel (Fig. 7). This indicates that membrane association of the distal I-II linker through its positive charges not only stabilizes a more reluctant channel state (Fig. 6) but also weakens inhibition of channel activity by phosphoinositide depletion.
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.