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A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

Kaur G, Pinggera A, Ortner NJ, Lieb A, Sinnegger-Brauns MJ, Yarov-Yarovoy V, Obermair GJ, Flucher BE, Striessnig J - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.

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All LTCC I-II linkers target to the plasma membrane in tsA-201 cells. Immunofluorescence images of anti-FLAG-labeled LTCC (CaV1.1–CaV1.4) I-II linkers and anti-V5-labeled palmitoylation-deficient β2a(C3S/C4Sβ2aV5) co-expressed in tsA-201 cells. A–D, left, anti-FLAG-labeled I-II linkers (I-II linkerFLAG); middle, anti-V5-labeled C3S/C4Sβ2a (C3S/C4Sβ2aV5); right, merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a). E, expression of only anti-FLAG-labeled distal 158 amino acids of the CaV1.3 α1 C terminus (C158FLAG), of anti-V5-labeled C3S/C4Sβ2aV5, and of anti-V5-labeled palmitoylated β2a (β2aV5), respectively. Representative cells from three independent experiments are shown. Scale bar, 10 μm.
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Figure 1: All LTCC I-II linkers target to the plasma membrane in tsA-201 cells. Immunofluorescence images of anti-FLAG-labeled LTCC (CaV1.1–CaV1.4) I-II linkers and anti-V5-labeled palmitoylation-deficient β2a(C3S/C4Sβ2aV5) co-expressed in tsA-201 cells. A–D, left, anti-FLAG-labeled I-II linkers (I-II linkerFLAG); middle, anti-V5-labeled C3S/C4Sβ2a (C3S/C4Sβ2aV5); right, merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a). E, expression of only anti-FLAG-labeled distal 158 amino acids of the CaV1.3 α1 C terminus (C158FLAG), of anti-V5-labeled C3S/C4Sβ2aV5, and of anti-V5-labeled palmitoylated β2a (β2aV5), respectively. Representative cells from three independent experiments are shown. Scale bar, 10 μm.

Mentions: To test whether plasma membrane binding is a property of all LTCC α1-subunits, we transfected FLAG-labeled I-II linkers of CaV1.1, CaV1.2, CaV1.3, and CaV1.4 α1-subunits (Fig. 1, A–D, left) into tsA-201 cells. Immunoblot analysis (not shown; n = 3) confirmed their expression as intact polypeptides. All LTCC I-II linkers were localized at the plasma membrane (Fig. 1, A–D). This localization pattern was independent of expression levels and indistinguishably observed in cells with weak and strong expression of the respective linkers (not shown). More than 85% of transfected cells (three independent experiments; 300 cells/experiment analyzed) showed this typical plasma membrane binding. In contrast, a FLAG-labeled control fragment derived from the CaV1.3 C terminus (FLAG-C158) clearly revealed a cytoplasmic distribution (Fig. 1E, first panel), as did the non-palmitoylated β2a mutant C3S/C4Sβ2a (Fig. 1E, middle). In contrast, normal palmitoylated β2a revealed the expected plasma membrane targeting (Fig. 1E, last panel) and thus served as a positive control. Consistent with our previous findings, C3S/C4Sβ2a (n = 3; Fig. 1, A–D, middle panel) and β3 (not shown, n = 3) (49) also were localized at the plasma membrane after co-expression with one of the four LTCC I-II linkers. This shows that all LTCC I-II linkers support β-subunit plasma membrane targeting.


A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

Kaur G, Pinggera A, Ortner NJ, Lieb A, Sinnegger-Brauns MJ, Yarov-Yarovoy V, Obermair GJ, Flucher BE, Striessnig J - J. Biol. Chem. (2015)

All LTCC I-II linkers target to the plasma membrane in tsA-201 cells. Immunofluorescence images of anti-FLAG-labeled LTCC (CaV1.1–CaV1.4) I-II linkers and anti-V5-labeled palmitoylation-deficient β2a(C3S/C4Sβ2aV5) co-expressed in tsA-201 cells. A–D, left, anti-FLAG-labeled I-II linkers (I-II linkerFLAG); middle, anti-V5-labeled C3S/C4Sβ2a (C3S/C4Sβ2aV5); right, merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a). E, expression of only anti-FLAG-labeled distal 158 amino acids of the CaV1.3 α1 C terminus (C158FLAG), of anti-V5-labeled C3S/C4Sβ2aV5, and of anti-V5-labeled palmitoylated β2a (β2aV5), respectively. Representative cells from three independent experiments are shown. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: All LTCC I-II linkers target to the plasma membrane in tsA-201 cells. Immunofluorescence images of anti-FLAG-labeled LTCC (CaV1.1–CaV1.4) I-II linkers and anti-V5-labeled palmitoylation-deficient β2a(C3S/C4Sβ2aV5) co-expressed in tsA-201 cells. A–D, left, anti-FLAG-labeled I-II linkers (I-II linkerFLAG); middle, anti-V5-labeled C3S/C4Sβ2a (C3S/C4Sβ2aV5); right, merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a). E, expression of only anti-FLAG-labeled distal 158 amino acids of the CaV1.3 α1 C terminus (C158FLAG), of anti-V5-labeled C3S/C4Sβ2aV5, and of anti-V5-labeled palmitoylated β2a (β2aV5), respectively. Representative cells from three independent experiments are shown. Scale bar, 10 μm.
Mentions: To test whether plasma membrane binding is a property of all LTCC α1-subunits, we transfected FLAG-labeled I-II linkers of CaV1.1, CaV1.2, CaV1.3, and CaV1.4 α1-subunits (Fig. 1, A–D, left) into tsA-201 cells. Immunoblot analysis (not shown; n = 3) confirmed their expression as intact polypeptides. All LTCC I-II linkers were localized at the plasma membrane (Fig. 1, A–D). This localization pattern was independent of expression levels and indistinguishably observed in cells with weak and strong expression of the respective linkers (not shown). More than 85% of transfected cells (three independent experiments; 300 cells/experiment analyzed) showed this typical plasma membrane binding. In contrast, a FLAG-labeled control fragment derived from the CaV1.3 C terminus (FLAG-C158) clearly revealed a cytoplasmic distribution (Fig. 1E, first panel), as did the non-palmitoylated β2a mutant C3S/C4Sβ2a (Fig. 1E, middle). In contrast, normal palmitoylated β2a revealed the expected plasma membrane targeting (Fig. 1E, last panel) and thus served as a positive control. Consistent with our previous findings, C3S/C4Sβ2a (n = 3; Fig. 1, A–D, middle panel) and β3 (not shown, n = 3) (49) also were localized at the plasma membrane after co-expression with one of the four LTCC I-II linkers. This shows that all LTCC I-II linkers support β-subunit plasma membrane targeting.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus