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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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Positively charged amino acids in region 536–544 are essential for CaV1.2 I-II linker targeting.A, immunofluorescence images of anti- FLAG-labeled deletion mutants as indicated and of GFP526–554. Staining of Δ536–554 was always intracellular but with a granular appearance. This was only very rarely seen with Δ526–554, which was typically of uniform distribution (cf. Fig. 2B). B, helical wheel representation of the proposed α-helix with positive charges indicated in blue. The 4 arginines important for membrane translocation are highlighted in blue with a yellow outline (A and B). C, schematic representation of positive residues (R and K) present in 526–554 sequence (CaV1.2 I-II linker). The four important arginines (Arg-537, Arg-538, Arg-541, and Arg-544) in amino acid sequence insert Δ544–554 and mutations (4R4A and 4R4E) in the CaV1.2 I-II linker are indicated. GFP-tagged 526–554 is also shown. Red, deletion in 544–554; yellow, 536–544; gray, α1-β subunit interaction site. D, putative phosphoinositide-binding domains of all LTCC I-II linkers are compared with published phosphoinositide-binding domains of Rit and K-Ras (22). All basic residues are marked in yellow, and ones that bind phosphoinositides are highlighted in red. E, immunofluorescence images of anti-FLAG-labeled charge neutralization linker mutants I-II 4R/4A and I-II 4R/4E (left), anti-V5-labeled C3S/C4Sβ2a (middle), and merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a) expressed in tsA-201 cells. Representative images from three independent experiments are shown. Scale bar, 10 μm.
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Figure 3: Positively charged amino acids in region 536–544 are essential for CaV1.2 I-II linker targeting.A, immunofluorescence images of anti- FLAG-labeled deletion mutants as indicated and of GFP526–554. Staining of Δ536–554 was always intracellular but with a granular appearance. This was only very rarely seen with Δ526–554, which was typically of uniform distribution (cf. Fig. 2B). B, helical wheel representation of the proposed α-helix with positive charges indicated in blue. The 4 arginines important for membrane translocation are highlighted in blue with a yellow outline (A and B). C, schematic representation of positive residues (R and K) present in 526–554 sequence (CaV1.2 I-II linker). The four important arginines (Arg-537, Arg-538, Arg-541, and Arg-544) in amino acid sequence insert Δ544–554 and mutations (4R4A and 4R4E) in the CaV1.2 I-II linker are indicated. GFP-tagged 526–554 is also shown. Red, deletion in 544–554; yellow, 536–544; gray, α1-β subunit interaction site. D, putative phosphoinositide-binding domains of all LTCC I-II linkers are compared with published phosphoinositide-binding domains of Rit and K-Ras (22). All basic residues are marked in yellow, and ones that bind phosphoinositides are highlighted in red. E, immunofluorescence images of anti-FLAG-labeled charge neutralization linker mutants I-II 4R/4A and I-II 4R/4E (left), anti-V5-labeled C3S/C4Sβ2a (middle), and merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a) expressed in tsA-201 cells. Representative images from three independent experiments are shown. Scale bar, 10 μm.

Mentions: To further identify the structural motif within the linker responsible for membrane targeting we expressed triple-FLAG-labeled linker peptides with deletions of equal size (29–30 amino acids) located in different parts of the CaV1.2 linker (Fig. 2A). The I-II linker of CaV1.2 was chosen for this analysis because it provided the best signal/noise ratio (not shown). Whereas membrane binding of mutants Δ436–465, Δ466–495, and Δ496–525 remained unaffected by the deletions (Fig. 2B), removal of amino acids 526–554 (Fig. 2, A and B; Δ526–554) abolished plasma membrane binding and resulted in cytoplasmic staining. Accordingly, mutants Δ436–465 and Δ496–525 translocated co-expressed C3S/C4Sβ2a to the plasma membrane (Fig. 2B), whereas Δ526–554 did not (Fig. 2B). Mutant Δ466–495 failed to target C3S/C4Sβ2a to the plasma membrane (Fig. 2B). This was expected, because the β-subunit α1-subunit interaction domain (AID) is also removed by this deletion. By creating additional deletion mutants Δ536–554 (no membrane binding; Fig. 3A) and Δ544–554 (membrane-bound; Fig. 3A) we found that essential structural determinants for membrane targeting must be localized within positions 536–543 (Fig. 2A).


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)

Positively charged amino acids in region 536–544 are essential for CaV1.2 I-II linker targeting.A, immunofluorescence images of anti- FLAG-labeled deletion mutants as indicated and of GFP526–554. Staining of Δ536–554 was always intracellular but with a granular appearance. This was only very rarely seen with Δ526–554, which was typically of uniform distribution (cf. Fig. 2B). B, helical wheel representation of the proposed α-helix with positive charges indicated in blue. The 4 arginines important for membrane translocation are highlighted in blue with a yellow outline (A and B). C, schematic representation of positive residues (R and K) present in 526–554 sequence (CaV1.2 I-II linker). The four important arginines (Arg-537, Arg-538, Arg-541, and Arg-544) in amino acid sequence insert Δ544–554 and mutations (4R4A and 4R4E) in the CaV1.2 I-II linker are indicated. GFP-tagged 526–554 is also shown. Red, deletion in 544–554; yellow, 536–544; gray, α1-β subunit interaction site. D, putative phosphoinositide-binding domains of all LTCC I-II linkers are compared with published phosphoinositide-binding domains of Rit and K-Ras (22). All basic residues are marked in yellow, and ones that bind phosphoinositides are highlighted in red. E, immunofluorescence images of anti-FLAG-labeled charge neutralization linker mutants I-II 4R/4A and I-II 4R/4E (left), anti-V5-labeled C3S/C4Sβ2a (middle), and merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a) expressed in tsA-201 cells. Representative images from three independent experiments are shown. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Positively charged amino acids in region 536–544 are essential for CaV1.2 I-II linker targeting.A, immunofluorescence images of anti- FLAG-labeled deletion mutants as indicated and of GFP526–554. Staining of Δ536–554 was always intracellular but with a granular appearance. This was only very rarely seen with Δ526–554, which was typically of uniform distribution (cf. Fig. 2B). B, helical wheel representation of the proposed α-helix with positive charges indicated in blue. The 4 arginines important for membrane translocation are highlighted in blue with a yellow outline (A and B). C, schematic representation of positive residues (R and K) present in 526–554 sequence (CaV1.2 I-II linker). The four important arginines (Arg-537, Arg-538, Arg-541, and Arg-544) in amino acid sequence insert Δ544–554 and mutations (4R4A and 4R4E) in the CaV1.2 I-II linker are indicated. GFP-tagged 526–554 is also shown. Red, deletion in 544–554; yellow, 536–544; gray, α1-β subunit interaction site. D, putative phosphoinositide-binding domains of all LTCC I-II linkers are compared with published phosphoinositide-binding domains of Rit and K-Ras (22). All basic residues are marked in yellow, and ones that bind phosphoinositides are highlighted in red. E, immunofluorescence images of anti-FLAG-labeled charge neutralization linker mutants I-II 4R/4A and I-II 4R/4E (left), anti-V5-labeled C3S/C4Sβ2a (middle), and merged images (green, anti-FLAG-labeled I-II linkers; red, anti-V5-labeled C3S/C4Sβ2a) expressed in tsA-201 cells. Representative images from three independent experiments are shown. Scale bar, 10 μm.
Mentions: To further identify the structural motif within the linker responsible for membrane targeting we expressed triple-FLAG-labeled linker peptides with deletions of equal size (29–30 amino acids) located in different parts of the CaV1.2 linker (Fig. 2A). The I-II linker of CaV1.2 was chosen for this analysis because it provided the best signal/noise ratio (not shown). Whereas membrane binding of mutants Δ436–465, Δ466–495, and Δ496–525 remained unaffected by the deletions (Fig. 2B), removal of amino acids 526–554 (Fig. 2, A and B; Δ526–554) abolished plasma membrane binding and resulted in cytoplasmic staining. Accordingly, mutants Δ436–465 and Δ496–525 translocated co-expressed C3S/C4Sβ2a to the plasma membrane (Fig. 2B), whereas Δ526–554 did not (Fig. 2B). Mutant Δ466–495 failed to target C3S/C4Sβ2a to the plasma membrane (Fig. 2B). This was expected, because the β-subunit α1-subunit interaction domain (AID) is also removed by this deletion. By creating additional deletion mutants Δ536–554 (no membrane binding; Fig. 3A) and Δ544–554 (membrane-bound; Fig. 3A) we found that essential structural determinants for membrane targeting must be localized within positions 536–543 (Fig. 2A).

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