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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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Reversal of I-II linker-induced plasma membrane targeting due to phosphoinositide depletion accompanied by increased intracellular Ca2+ levels.A, mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells treated with the PLC activator m-3M3FBS (50 μm) together with wortmannin (20 μm); left, mRFP-labeled PH domain of PLCδ; right, GFP-labeled 526–554. Representative cells from three independent experiments are shown. B and C, different constructs were expressed in tsA-201 cells and subsequently treated with the PLC activator m-3M3FBS (50 μm) and additional ionomycin (5 μm). B, left, mRFP-labeled PH-PLCδ; middle, GFP-labeled β3-subunit co-expressed with CaV1.2 I-II linker; right, GFP-labeled peptide 526–554. The membrane-localized staining reversed by treatment is indicated by an arrow. Note that in the majority of cells, GFP526–554 also showed strong nuclear targeting (for an explanation, see “Results”), which was absent in dividing cells without distinct nuclei (Figs. 3A and 5A). C, GFP-labeled β2a alone. To quantify the relocation from the membrane, pixel intensity from three representative areas of interest within the membrane of each cell were background-corrected, and the membrane/cytoplasm ratio was calculated from the means obtained before (blue) and after (red) PLC activation. Means ± S.E. (error bars) are shown for the indicated number of cells. Paired Student's t test was used. **, p = 0.0049 (PH-PLCδ, m-3M3FBS + wortmannin, n = 3); ***, p = 0.00016 (peptide 526–554, m-3M3FBS + wortmannin, n = 3); **, p = 0.00427 (PH-PLCδ, m-3M3FBS + ionomycin, n = 15); **, p = 0.01087 (β3-subunit + CaV1.2 I-II linker, m-3M3FBS + ionomycin, n = 5); ***, p = 0.0010 (peptide 526–554, m-3M3FBS + ionomycin, n = 3); p = 0.81799 (β2a, m-3M3FBS + ionomycin, n = 6). Scale bar, 10 μm.
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Figure 5: Reversal of I-II linker-induced plasma membrane targeting due to phosphoinositide depletion accompanied by increased intracellular Ca2+ levels.A, mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells treated with the PLC activator m-3M3FBS (50 μm) together with wortmannin (20 μm); left, mRFP-labeled PH domain of PLCδ; right, GFP-labeled 526–554. Representative cells from three independent experiments are shown. B and C, different constructs were expressed in tsA-201 cells and subsequently treated with the PLC activator m-3M3FBS (50 μm) and additional ionomycin (5 μm). B, left, mRFP-labeled PH-PLCδ; middle, GFP-labeled β3-subunit co-expressed with CaV1.2 I-II linker; right, GFP-labeled peptide 526–554. The membrane-localized staining reversed by treatment is indicated by an arrow. Note that in the majority of cells, GFP526–554 also showed strong nuclear targeting (for an explanation, see “Results”), which was absent in dividing cells without distinct nuclei (Figs. 3A and 5A). C, GFP-labeled β2a alone. To quantify the relocation from the membrane, pixel intensity from three representative areas of interest within the membrane of each cell were background-corrected, and the membrane/cytoplasm ratio was calculated from the means obtained before (blue) and after (red) PLC activation. Means ± S.E. (error bars) are shown for the indicated number of cells. Paired Student's t test was used. **, p = 0.0049 (PH-PLCδ, m-3M3FBS + wortmannin, n = 3); ***, p = 0.00016 (peptide 526–554, m-3M3FBS + wortmannin, n = 3); **, p = 0.00427 (PH-PLCδ, m-3M3FBS + ionomycin, n = 15); **, p = 0.01087 (β3-subunit + CaV1.2 I-II linker, m-3M3FBS + ionomycin, n = 5); ***, p = 0.0010 (peptide 526–554, m-3M3FBS + ionomycin, n = 3); p = 0.81799 (β2a, m-3M3FBS + ionomycin, n = 6). 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)

Reversal of I-II linker-induced plasma membrane targeting due to phosphoinositide depletion accompanied by increased intracellular Ca2+ levels.A, mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells treated with the PLC activator m-3M3FBS (50 μm) together with wortmannin (20 μm); left, mRFP-labeled PH domain of PLCδ; right, GFP-labeled 526–554. Representative cells from three independent experiments are shown. B and C, different constructs were expressed in tsA-201 cells and subsequently treated with the PLC activator m-3M3FBS (50 μm) and additional ionomycin (5 μm). B, left, mRFP-labeled PH-PLCδ; middle, GFP-labeled β3-subunit co-expressed with CaV1.2 I-II linker; right, GFP-labeled peptide 526–554. The membrane-localized staining reversed by treatment is indicated by an arrow. Note that in the majority of cells, GFP526–554 also showed strong nuclear targeting (for an explanation, see “Results”), which was absent in dividing cells without distinct nuclei (Figs. 3A and 5A). C, GFP-labeled β2a alone. To quantify the relocation from the membrane, pixel intensity from three representative areas of interest within the membrane of each cell were background-corrected, and the membrane/cytoplasm ratio was calculated from the means obtained before (blue) and after (red) PLC activation. Means ± S.E. (error bars) are shown for the indicated number of cells. Paired Student's t test was used. **, p = 0.0049 (PH-PLCδ, m-3M3FBS + wortmannin, n = 3); ***, p = 0.00016 (peptide 526–554, m-3M3FBS + wortmannin, n = 3); **, p = 0.00427 (PH-PLCδ, m-3M3FBS + ionomycin, n = 15); **, p = 0.01087 (β3-subunit + CaV1.2 I-II linker, m-3M3FBS + ionomycin, n = 5); ***, p = 0.0010 (peptide 526–554, m-3M3FBS + ionomycin, n = 3); p = 0.81799 (β2a, m-3M3FBS + ionomycin, n = 6). Scale bar, 10 μm.
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Figure 5: Reversal of I-II linker-induced plasma membrane targeting due to phosphoinositide depletion accompanied by increased intracellular Ca2+ levels.A, mRFP-labeled PH-PLCδ and GFP-labeled 526–554 were co-expressed with untagged muscarinic M1 receptor in tsA-201 cells treated with the PLC activator m-3M3FBS (50 μm) together with wortmannin (20 μm); left, mRFP-labeled PH domain of PLCδ; right, GFP-labeled 526–554. Representative cells from three independent experiments are shown. B and C, different constructs were expressed in tsA-201 cells and subsequently treated with the PLC activator m-3M3FBS (50 μm) and additional ionomycin (5 μm). B, left, mRFP-labeled PH-PLCδ; middle, GFP-labeled β3-subunit co-expressed with CaV1.2 I-II linker; right, GFP-labeled peptide 526–554. The membrane-localized staining reversed by treatment is indicated by an arrow. Note that in the majority of cells, GFP526–554 also showed strong nuclear targeting (for an explanation, see “Results”), which was absent in dividing cells without distinct nuclei (Figs. 3A and 5A). C, GFP-labeled β2a alone. To quantify the relocation from the membrane, pixel intensity from three representative areas of interest within the membrane of each cell were background-corrected, and the membrane/cytoplasm ratio was calculated from the means obtained before (blue) and after (red) PLC activation. Means ± S.E. (error bars) are shown for the indicated number of cells. Paired Student's t test was used. **, p = 0.0049 (PH-PLCδ, m-3M3FBS + wortmannin, n = 3); ***, p = 0.00016 (peptide 526–554, m-3M3FBS + wortmannin, n = 3); **, p = 0.00427 (PH-PLCδ, m-3M3FBS + ionomycin, n = 15); **, p = 0.01087 (β3-subunit + CaV1.2 I-II linker, m-3M3FBS + ionomycin, n = 5); ***, p = 0.0010 (peptide 526–554, m-3M3FBS + ionomycin, n = 3); p = 0.81799 (β2a, m-3M3FBS + ionomycin, n = 6). 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