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Dynamic phospholipid interaction of ?2e subunit regulates the gating of voltage-gated Ca2+ channels.

ABSTRACT: High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming ?1 and auxiliary ? and ?2? subunits. The subcellular localization and membrane interactions of the ? subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The ?2 isoform ?2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric ? subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of ?2e and anionic phospholipids was sufficient for ?2e membrane targeting. Binding of the ?2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for ?2e targeting to the plasma membrane. Membrane binding of the ?2e subunit slowed Ca(V)2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca(V)2.2(?2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of ?2e subunits from membrane to cytosol, but not that of ?2a or ?3, resulting in fast inactivation of Ca(V)2.2 channels with ?2e. These results suggest that membrane targeting of the ?2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of ?2e with membrane PIs results in functional changes in Ca(V) channel gating. The phospholipid-protein interaction observed here provides structural insight into mechanisms of membrane-protein association and the role of phospholipids in ion channel regulation.

SUBMITTER: Kim DI

PROVIDER: S-EPMC4442786 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Dynamic phospholipid interaction of β2e subunit regulates the gating of voltage-gated Ca2+ channels.

Kim Dong-Il DI Park Yongsoo Y Jang Deok-Jin DJ Suh Byung-Chang BC

The Journal of general physiology 20150511 6

High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Usin ...[more]

PMID: 25964431

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Project description:Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III-IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.

Dataset Information

Dynamic phospholipid interaction of ?2e subunit regulates the gating of voltage-gated Ca2+ channels.

Publications

Dynamic phospholipid interaction of β2e subunit regulates the gating of voltage-gated Ca2+ channels.

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