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Membrane-associated guanylate kinase-like properties of beta-subunits required for modulation of voltage-dependent Ca2+ channels.

ABSTRACT: High-voltage-activated Ca2+ channels regulate diverse functions ranging from muscle contraction to synaptic transmission. Association between auxiliary beta- and distinct pore-forming alpha1-subunits is obligatory for forming functional high-voltage-activated Ca2+ channels, yet the structural determinants underlying this interaction remain poorly understood. Recently, homology modeling of Ca(2+)-channel beta1b-subunit identified src homology 3 (SH3) and guanylate kinase (GK) motifs in a tandem arrangement reminiscent of the membrane-associated guanylate kinase (MAGUK) class of scaffolding proteins. However, direct evidence for MAGUK-like properties and their functional implications in beta-subunits is lacking. Here, we show a functional requirement for both SH3 and GK domains in beta2a. Point mutations in either the putative beta2a SH3 or GK domains severely blunted modulation of recombinant L-type channels, showing the importance of both motifs for a functional alpha1-beta interaction. Coexpression of these functionally deficient beta2a-SH3 and GK mutants rescued WT currents, demonstrating trans complementation similar to that observed in MAGUKs. Truncated "hemi-beta2a" subunits, containing either the SH3 or GK domain, were ineffective on their own, but reconstituted WT currents when coexpressed. Moreover, the SH3 and GK domains were found to interact in vitro. These findings reveal MAGUK-like properties in beta-subunits that are critical for alpha1-subunit modulation, revise current models of alpha1-beta association, and predict new physiological dimensions of beta-subunit function.

SUBMITTER: Takahashi SX

PROVIDER: S-EPMC406488 | biostudies-other | 2004 May

REPOSITORIES: biostudies-other

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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.

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Membrane-associated guanylate kinase-like properties of beta-subunits required for modulation of voltage-dependent Ca2+ channels.

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