A polybasic plasma membrane binding motif in the I-II linker stabilizes voltage-gated Ca<inf>V</inf>1.2 calcium channel function

Gurjot Kaur, Alexandra Pinggera, Nadine J. Ortner, Andreas Lieb, Martina J. Sinnegger-Brauns, Vladimir Yarov-Yarovoy, Gerald J. Obermair, Bernhard E. Flucher, Jörg Striessnig

Research output: Contribution to journalArticle

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Abstract

L-type voltage-gated Ca<sup>2+</sup> channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The poreforming α<inf>1</inf>-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Ca<inf>v</inf>1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphospho-inositide breakdown, and was accelerated by elevated intracellular Ca<sup>2+</sup> levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Ca<inf>v</inf>1.2 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 α<inf>1</inf>-subunits essential for stabilizing normal Ca<sup>2+</sup> channel function.

Original languageEnglish (US)
Pages (from-to)21086-21100
Number of pages15
JournalJournal of Biological Chemistry
Volume290
Issue number34
DOIs
StatePublished - Aug 21 2015

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Cell membranes
Cell Membrane
Electric potential
Type C Phospholipases
Arginine
Chemical activation
Electrophysiology
Membranes
Phosphatidylinositol Phosphates
Molecular modeling
Clamping devices
Muscle Contraction
Ion Channels
Fluorescence Microscopy
Gene expression
Muscle
Conformations
L-type calcium channel alpha(1C)
Phospholipids
Microscopic examination

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

A polybasic plasma membrane binding motif in the I-II linker stabilizes voltage-gated Ca<inf>V</inf>1.2 calcium channel function. / Kaur, Gurjot; Pinggera, Alexandra; Ortner, Nadine J.; Lieb, Andreas; Sinnegger-Brauns, Martina J.; Yarov-Yarovoy, Vladimir; Obermair, Gerald J.; Flucher, Bernhard E.; Striessnig, Jörg.

In: Journal of Biological Chemistry, Vol. 290, No. 34, 21.08.2015, p. 21086-21100.

Research output: Contribution to journalArticle

Kaur, G, Pinggera, A, Ortner, NJ, Lieb, A, Sinnegger-Brauns, MJ, Yarov-Yarovoy, V, Obermair, GJ, Flucher, BE & Striessnig, J 2015, 'A polybasic plasma membrane binding motif in the I-II linker stabilizes voltage-gated Ca<inf>V</inf>1.2 calcium channel function', Journal of Biological Chemistry, vol. 290, no. 34, pp. 21086-21100. https://doi.org/10.1074/jbc.M115.645671
Kaur, Gurjot ; Pinggera, Alexandra ; Ortner, Nadine J. ; Lieb, Andreas ; Sinnegger-Brauns, Martina J. ; Yarov-Yarovoy, Vladimir ; Obermair, Gerald J. ; Flucher, Bernhard E. ; Striessnig, Jörg. / A polybasic plasma membrane binding motif in the I-II linker stabilizes voltage-gated Ca<inf>V</inf>1.2 calcium channel function. In: Journal of Biological Chemistry. 2015 ; Vol. 290, No. 34. pp. 21086-21100.
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AU - Kaur, Gurjot

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AU - Ortner, Nadine J.

AU - Lieb, Andreas

AU - Sinnegger-Brauns, Martina J.

AU - Yarov-Yarovoy, Vladimir

AU - Obermair, Gerald J.

AU - Flucher, Bernhard E.

AU - Striessnig, Jörg

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AB - L-type voltage-gated Ca2+ channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The poreforming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphospho-inositide breakdown, and was accelerated by elevated intracellular Ca2+ levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. 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 Ca2+ channel function.

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