title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.
abstract: L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. 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.
authors.affiliation: Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Gurjot
authors.initials: G
authors.lastname: Kaur
authors.affiliation: Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Pinggera
authors.affiliation: Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Nadine J
authors.initials: NJ
authors.lastname: Ortner
authors.affiliation: Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Lieb
authors.affiliation: Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Martina J
authors.initials: MJ
authors.lastname: Sinnegger-Brauns
authors.affiliation: Department of Physiology and Membrane Biology, UC Davis School of Medicine, Davis, California 95616.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Yarov-Yarovoy
authors.affiliation: Division of Physiology, Medical University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Obermair
authors.affiliation: Division of Physiology, Medical University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Bernhard E
authors.initials: BE
authors.lastname: Flucher
authors.affiliation: Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria. Electronic address: joerg.striessnig@uibk.ac.at.
authors.forename: Jörg
authors.initials: J
authors.lastname: Striessnig
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D017136:Ion Transport; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism; D020864:Calcium Channels, N-Type
chemical_list: D002118:Calcium; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; C105656:CACNA1A protein, human; D020864:Calcium Channels, N-Type; C412223:CACNA1S protein, human; C419385:L-type calcium channel alpha(1C)
keywords: Cav1.2; L-type calcium channels; calcium channel; cytoplasmic domain; electrophysiology; phospholipid; plasma membrane; protein motif