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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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Plasma membrane interaction of CaV1.2 I-II linker is not influenced by muscarinic receptor activation or rapamycin induced PIP or PIP2 depletion.A and B, mRFP-labeled PH-PLCδ and GFP-labeled β3 with untagged CaV1.2 I-II linker or mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells and subsequently treated with the M1 receptor agonist Oxo-M (10 μm). Fluorescence was visualized using live cell imaging, and images before (t = 0 s) and after (t = 5 s) the drug application were recorded. A, left, mRFP-labeled PH-PLCδ; right, GFP-labeled β3 in the same cells co-expressed with untagged CaV1.2 I-II linker and mRFP-labeled PH-PLCδ. B, left, mRFP-labeled PH-PLCδ; right, GFP-labeled peptide 526–554 in the same cells co-expressed with mRFP-labeled PH-PLCδ. Representative cells from three independent experiments are shown. C–F, cells co-transfected with Lyn11-FRB, RFP-pseudojanin, and GFP-PH-Osh2x2 or CaV1.2 I-II linker together with β3-GFP. Fluorescence was visualized before (t = 0 s) and after drug (t = 60 s) application. C and D, RFP-labeled pseudojanin (left) with GFP-labeled PH-Osh2x2 (right) as control before and after rapamycin treatment. Representative cells from eight independent experiments are shown. E, left, RFP-labeled pseudojanin; right, GFP-labeled β3, before and after rapamycin application. Representative cells from three independent experiments are shown. F, left, RFP-labeled pseudojanin; right, GFP-labeled β3 preincubated with wortmannin (before) and after the addition of rapamycin (after). Representative cells from three independent experiments are shown. Scale bar, 10 μm.
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Figure 4: Plasma membrane interaction of CaV1.2 I-II linker is not influenced by muscarinic receptor activation or rapamycin induced PIP or PIP2 depletion.A and B, mRFP-labeled PH-PLCδ and GFP-labeled β3 with untagged CaV1.2 I-II linker or mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells and subsequently treated with the M1 receptor agonist Oxo-M (10 μm). Fluorescence was visualized using live cell imaging, and images before (t = 0 s) and after (t = 5 s) the drug application were recorded. A, left, mRFP-labeled PH-PLCδ; right, GFP-labeled β3 in the same cells co-expressed with untagged CaV1.2 I-II linker and mRFP-labeled PH-PLCδ. B, left, mRFP-labeled PH-PLCδ; right, GFP-labeled peptide 526–554 in the same cells co-expressed with mRFP-labeled PH-PLCδ. Representative cells from three independent experiments are shown. C–F, cells co-transfected with Lyn11-FRB, RFP-pseudojanin, and GFP-PH-Osh2x2 or CaV1.2 I-II linker together with β3-GFP. Fluorescence was visualized before (t = 0 s) and after drug (t = 60 s) application. C and D, RFP-labeled pseudojanin (left) with GFP-labeled PH-Osh2x2 (right) as control before and after rapamycin treatment. Representative cells from eight independent experiments are shown. E, left, RFP-labeled pseudojanin; right, GFP-labeled β3, before and after rapamycin application. Representative cells from three independent experiments are shown. F, left, RFP-labeled pseudojanin; right, GFP-labeled β3 preincubated with wortmannin (before) and after the addition of rapamycin (after). Representative cells from three independent experiments are shown. Scale bar, 10 μm.

Mentions: To assess whether this polybasic motif is sufficient to induce plasma membrane binding when attached to an otherwise cytoplasmic protein, we fused residues 526–554 to the C terminus of GFP (GFP526–554; Fig. 3, A and C). This construct localized to the plasma membrane (Fig. 3A). It also accumulated in the nucleus of the vast majority of transfected cells. Notably, a distinct feature of lipid-interacting polybasic membrane targeting motifs is their similarity to nuclear localization sequences (22), a property that may also account for the nuclear targeting of GFP526–554. However, nuclear staining did not obscure plasma membrane binding (see also Figs. 4A and 5B), and this was also confirmed in dividing cells in which nuclear staining was completely absent (Fig. 3A, GFP526–554, right panel; see also Fig. 5A). Taken together, these findings clearly demonstrate that positive charges at the C-terminal end of the CaV1.2 I-II linker form a plasma membrane binding motif sufficient for translocating the cytoplasmic I-II linker and fused GFP to the plasma membrane.


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)

Plasma membrane interaction of CaV1.2 I-II linker is not influenced by muscarinic receptor activation or rapamycin induced PIP or PIP2 depletion.A and B, mRFP-labeled PH-PLCδ and GFP-labeled β3 with untagged CaV1.2 I-II linker or mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells and subsequently treated with the M1 receptor agonist Oxo-M (10 μm). Fluorescence was visualized using live cell imaging, and images before (t = 0 s) and after (t = 5 s) the drug application were recorded. A, left, mRFP-labeled PH-PLCδ; right, GFP-labeled β3 in the same cells co-expressed with untagged CaV1.2 I-II linker and mRFP-labeled PH-PLCδ. B, left, mRFP-labeled PH-PLCδ; right, GFP-labeled peptide 526–554 in the same cells co-expressed with mRFP-labeled PH-PLCδ. Representative cells from three independent experiments are shown. C–F, cells co-transfected with Lyn11-FRB, RFP-pseudojanin, and GFP-PH-Osh2x2 or CaV1.2 I-II linker together with β3-GFP. Fluorescence was visualized before (t = 0 s) and after drug (t = 60 s) application. C and D, RFP-labeled pseudojanin (left) with GFP-labeled PH-Osh2x2 (right) as control before and after rapamycin treatment. Representative cells from eight independent experiments are shown. E, left, RFP-labeled pseudojanin; right, GFP-labeled β3, before and after rapamycin application. Representative cells from three independent experiments are shown. F, left, RFP-labeled pseudojanin; right, GFP-labeled β3 preincubated with wortmannin (before) and after the addition of rapamycin (after). Representative cells from three independent experiments are shown. Scale bar, 10 μm.
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Figure 4: Plasma membrane interaction of CaV1.2 I-II linker is not influenced by muscarinic receptor activation or rapamycin induced PIP or PIP2 depletion.A and B, mRFP-labeled PH-PLCδ and GFP-labeled β3 with untagged CaV1.2 I-II linker or mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells and subsequently treated with the M1 receptor agonist Oxo-M (10 μm). Fluorescence was visualized using live cell imaging, and images before (t = 0 s) and after (t = 5 s) the drug application were recorded. A, left, mRFP-labeled PH-PLCδ; right, GFP-labeled β3 in the same cells co-expressed with untagged CaV1.2 I-II linker and mRFP-labeled PH-PLCδ. B, left, mRFP-labeled PH-PLCδ; right, GFP-labeled peptide 526–554 in the same cells co-expressed with mRFP-labeled PH-PLCδ. Representative cells from three independent experiments are shown. C–F, cells co-transfected with Lyn11-FRB, RFP-pseudojanin, and GFP-PH-Osh2x2 or CaV1.2 I-II linker together with β3-GFP. Fluorescence was visualized before (t = 0 s) and after drug (t = 60 s) application. C and D, RFP-labeled pseudojanin (left) with GFP-labeled PH-Osh2x2 (right) as control before and after rapamycin treatment. Representative cells from eight independent experiments are shown. E, left, RFP-labeled pseudojanin; right, GFP-labeled β3, before and after rapamycin application. Representative cells from three independent experiments are shown. F, left, RFP-labeled pseudojanin; right, GFP-labeled β3 preincubated with wortmannin (before) and after the addition of rapamycin (after). Representative cells from three independent experiments are shown. Scale bar, 10 μm.
Mentions: To assess whether this polybasic motif is sufficient to induce plasma membrane binding when attached to an otherwise cytoplasmic protein, we fused residues 526–554 to the C terminus of GFP (GFP526–554; Fig. 3, A and C). This construct localized to the plasma membrane (Fig. 3A). It also accumulated in the nucleus of the vast majority of transfected cells. Notably, a distinct feature of lipid-interacting polybasic membrane targeting motifs is their similarity to nuclear localization sequences (22), a property that may also account for the nuclear targeting of GFP526–554. However, nuclear staining did not obscure plasma membrane binding (see also Figs. 4A and 5B), and this was also confirmed in dividing cells in which nuclear staining was completely absent (Fig. 3A, GFP526–554, right panel; see also Fig. 5A). Taken together, these findings clearly demonstrate that positive charges at the C-terminal end of the CaV1.2 I-II linker form a plasma membrane binding motif sufficient for translocating the cytoplasmic I-II linker and fused GFP to the plasma membrane.

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