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Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel ?4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

ABSTRACT: Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory ? subunits. All RGK proteins bind to voltage-gated Ca(2+) channel ? subunit (Cav?) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cav?4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the ?4 guanylate kinase domain contributing to a 156 ± 18 µM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca(2+) channel ?1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cav?4a, but were unaffected by Rem2 when coexpressed with a Cav?4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cav?4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.

SUBMITTER: Xu X

PROVIDER: S-EPMC4415010 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

Xu Xingfu X Zhang Fangxiong F Zamponi Gerald W GW Horne William A WA

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20150106 5

Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory β subunits. All RGK proteins bind to voltage-gated Ca(2+) channel β subunit (Cavβ) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cavβ4a and measure its binding affinity. Our experiments revealed ...[more]

PMID: 25563298

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Project description:Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules ?-neurexins (?Nrxns), the extracellularly longer variants of the three vertebrate Nrxn genes. Ca2+ influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (CaV2.1) triggers release of fusion-ready vesicles at many boutons. ?2? Auxiliary subunits regulate trafficking and kinetic properties of CaV2.1 pore-forming subunits but it has remained unclear if this involves ?Nrxns. Using live cell imaging with Ca2+ indicators, we report here that the total presynaptic Ca2+ influx in primary hippocampal neurons of ?Nrxn triple knock-out mice of both sexes is reduced and involved lower CaV2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of CaV2.1 pore-forming subunits, and elevated surface mobility of ?2?-1 on axons. Overexpression of Nrxn1? in ?Nrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca2+ influx and synaptic vesicle release. Moreover, coexpression of Nrxn1? together with ?2?-1 subunits facilitates Ca2+ influx further but causes little augmentation together with a different subunit, ?2?-3, suggesting remarkable specificity. Expression of defined recombinant CaV2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1? in combination with ?2?-1, but not with ?2?-3, facilitates Ca2+ currents of recombinant CaV2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1? acts as a positive regulator of Ca2+ influx through CaV2.1 channels containing ?2?-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENT Synaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their ?2?-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.

Dataset Information

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel ?4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

Publications

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

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