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: Homology, de novo, and full-atom modeling of the voltage-sensing domain (VSD) of native and mutant CaV1.2 channels was performed using the Rosetta membrane method (39–41) and the x-ray structure of the bacterial voltage-gated Na+ channel (NaVAb) VSD (42) as a template. Sequence alignment between native CaV1.2 and NaVAb VSDs shown in Fig. 8A was generated using the HHpred server (43, 44). The backbone structure of the transmembrane regions of CaV1.2 was built based on NaVAb VSD template. The 19-residue N-terminal region and S1-S2, S2-S3, and S3-S4 loops of CaV1.2 VSD were built de novo using the Rosetta cyclic coordinate descent loop modeling method (45) guided by membrane environment-specific energy function (39, 46). 10,000 models were generated for each CaV1.2 channel construct, and the top 10% of models ranked by total score were clustered (47) using root mean square deviation threshold that generates at least 150–200 models in the largest cluster. Models representing centers of the top five clusters and the best 10 models by total score were chosen for visual analysis. The top cluster and all 10 lowest energy models of native CaV1.2 showed very similar conformation of the domain I-II linker region (see Fig. 8). None of the top five clusters and 10 lowest energy models of alanine or glutamate mutants of CaV1.2 showed similar conformations of the domain I-II linker region (see Fig. 8). All structural figures were generated using the UCSF Chimera package (48).
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.