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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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Structural models of the domain I-II linker region of native CaV1.2 channel.A, sequence alignment between the native CaV1.2 I-II linker region and the domain II voltage-sensing domain (rCaV1.2-DII-VSD) and NaVAb voltage-sensing domain (NaVAb-VSD). Transmembrane segments S1–S4 are underlined by black bars and labeled. Amino acids were colored using the Zappo color scheme in Jalview. B–D, transmembrane view of the ribbon representation of the top cluster models of the VSD of CaV1.2 with the 10 lowest energy Rosetta models superimposed in B and space-filling representations of arginine side chains in the domain I-II linker helix in C and of large hydrophobic side chains in the I-II linker helix in D. E and F, transmembrane view of ribbon representation of the top five clusters and 10 lowest energy Rosetta models of the CaV1.2 VSD of alanine mutants superimposed in E and of glutamate mutants superimposed in F. Models are colored in a rainbow scheme from blue (N-terminal region before S1 segment) to red (S4 segment). Transmembrane segments S1–S4 are labeled accordingly. Black bars, approximate location of the extracellular and intracellular edges of the hydrophobic layer of the membrane.
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Figure 8: Structural models of the domain I-II linker region of native CaV1.2 channel.A, sequence alignment between the native CaV1.2 I-II linker region and the domain II voltage-sensing domain (rCaV1.2-DII-VSD) and NaVAb voltage-sensing domain (NaVAb-VSD). Transmembrane segments S1–S4 are underlined by black bars and labeled. Amino acids were colored using the Zappo color scheme in Jalview. B–D, transmembrane view of the ribbon representation of the top cluster models of the VSD of CaV1.2 with the 10 lowest energy Rosetta models superimposed in B and space-filling representations of arginine side chains in the domain I-II linker helix in C and of large hydrophobic side chains in the I-II linker helix in D. E and F, transmembrane view of ribbon representation of the top five clusters and 10 lowest energy Rosetta models of the CaV1.2 VSD of alanine mutants superimposed in E and of glutamate mutants superimposed in F. Models are colored in a rainbow scheme from blue (N-terminal region before S1 segment) to red (S4 segment). Transmembrane segments S1–S4 are labeled accordingly. Black bars, approximate location of the extracellular and intracellular edges of the hydrophobic layer of the membrane.

Mentions: Homology, de novo, and full-atom modeling of the voltage-sensing domain (VSD) of native and mutant CaV1.2 channels was performed using the Rosetta membrane method (39–41) and the x-ray structure of the bacterial voltage-gated Na+ channel (NaVAb) VSD (42) as a template. Sequence alignment between native CaV1.2 and NaVAb VSDs shown in Fig. 8A was generated using the HHpred server (43, 44). The backbone structure of the transmembrane regions of CaV1.2 was built based on NaVAb VSD template. The 19-residue N-terminal region and S1-S2, S2-S3, and S3-S4 loops of CaV1.2 VSD were built de novo using the Rosetta cyclic coordinate descent loop modeling method (45) guided by membrane environment-specific energy function (39, 46). 10,000 models were generated for each CaV1.2 channel construct, and the top 10% of models ranked by total score were clustered (47) using root mean square deviation threshold that generates at least 150–200 models in the largest cluster. Models representing centers of the top five clusters and the best 10 models by total score were chosen for visual analysis. The top cluster and all 10 lowest energy models of native CaV1.2 showed very similar conformation of the domain I-II linker region (see Fig. 8). None of the top five clusters and 10 lowest energy models of alanine or glutamate mutants of CaV1.2 showed similar conformations of the domain I-II linker region (see Fig. 8). All structural figures were generated using the UCSF Chimera package (48).


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)

Structural models of the domain I-II linker region of native CaV1.2 channel.A, sequence alignment between the native CaV1.2 I-II linker region and the domain II voltage-sensing domain (rCaV1.2-DII-VSD) and NaVAb voltage-sensing domain (NaVAb-VSD). Transmembrane segments S1–S4 are underlined by black bars and labeled. Amino acids were colored using the Zappo color scheme in Jalview. B–D, transmembrane view of the ribbon representation of the top cluster models of the VSD of CaV1.2 with the 10 lowest energy Rosetta models superimposed in B and space-filling representations of arginine side chains in the domain I-II linker helix in C and of large hydrophobic side chains in the I-II linker helix in D. E and F, transmembrane view of ribbon representation of the top five clusters and 10 lowest energy Rosetta models of the CaV1.2 VSD of alanine mutants superimposed in E and of glutamate mutants superimposed in F. Models are colored in a rainbow scheme from blue (N-terminal region before S1 segment) to red (S4 segment). Transmembrane segments S1–S4 are labeled accordingly. Black bars, approximate location of the extracellular and intracellular edges of the hydrophobic layer of the membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4543666&req=5

Figure 8: Structural models of the domain I-II linker region of native CaV1.2 channel.A, sequence alignment between the native CaV1.2 I-II linker region and the domain II voltage-sensing domain (rCaV1.2-DII-VSD) and NaVAb voltage-sensing domain (NaVAb-VSD). Transmembrane segments S1–S4 are underlined by black bars and labeled. Amino acids were colored using the Zappo color scheme in Jalview. B–D, transmembrane view of the ribbon representation of the top cluster models of the VSD of CaV1.2 with the 10 lowest energy Rosetta models superimposed in B and space-filling representations of arginine side chains in the domain I-II linker helix in C and of large hydrophobic side chains in the I-II linker helix in D. E and F, transmembrane view of ribbon representation of the top five clusters and 10 lowest energy Rosetta models of the CaV1.2 VSD of alanine mutants superimposed in E and of glutamate mutants superimposed in F. Models are colored in a rainbow scheme from blue (N-terminal region before S1 segment) to red (S4 segment). Transmembrane segments S1–S4 are labeled accordingly. Black bars, approximate location of the extracellular and intracellular edges of the hydrophobic layer of the membrane.
Mentions: Homology, de novo, and full-atom modeling of the voltage-sensing domain (VSD) of native and mutant CaV1.2 channels was performed using the Rosetta membrane method (39–41) and the x-ray structure of the bacterial voltage-gated Na+ channel (NaVAb) VSD (42) as a template. Sequence alignment between native CaV1.2 and NaVAb VSDs shown in Fig. 8A was generated using the HHpred server (43, 44). The backbone structure of the transmembrane regions of CaV1.2 was built based on NaVAb VSD template. The 19-residue N-terminal region and S1-S2, S2-S3, and S3-S4 loops of CaV1.2 VSD were built de novo using the Rosetta cyclic coordinate descent loop modeling method (45) guided by membrane environment-specific energy function (39, 46). 10,000 models were generated for each CaV1.2 channel construct, and the top 10% of models ranked by total score were clustered (47) using root mean square deviation threshold that generates at least 150–200 models in the largest cluster. Models representing centers of the top five clusters and the best 10 models by total score were chosen for visual analysis. The top cluster and all 10 lowest energy models of native CaV1.2 showed very similar conformation of the domain I-II linker region (see Fig. 8). None of the top five clusters and 10 lowest energy models of alanine or glutamate mutants of CaV1.2 showed similar conformations of the domain I-II linker region (see Fig. 8). All structural figures were generated using the UCSF Chimera package (48).

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