Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.DOI: http://dx.doi.org/10.7554/eLife.13662.001

Cardiac tubular-like membrane invaginations contain the membrane scaffolding protein BIN1, which tethers dynamic microtubules that deliver calcium channels directly to T-tubule membrane.

Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)‐fused auxiliary Ca2+ channel β subunits (β1‐β4) in cultured adult rat heart cells, to explore new dimensions of β subunit functions in vivo. Distinct β‐GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All β‐GFP subunits increased the native cardiac whole‐cell L‐type Ca2+ channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole‐cell current density was non‐uniform between β subunits, with a rank order of potency β2aαβ4 > β1b > β3. For each β subunit, the increase in L‐type current density was accompanied by a correlative increase in the maximal gating charge (Qmax) moved with depolarization. However, β subunits produced characteristic effects on single L‐type channel gating, resulting in divergent effects on channel open probability (Po). Quantitative analysis and modelling of single‐channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L‐type channel β subunit, we cloned a new rat β2 splice variant, β2b, from heart using 5′ rapid amplification of cDNA ends (RACE) PCR. By contrast with β2a, expression of β2b in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into β subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the β subunit component of cardiac L‐type Ca2+ channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.

Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 channels 1-4. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Ca V 1.2 α 1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca 2+ channels. We expressed α 1C or β 2B subunits conjugated to ascorbate-peroxidase 5 in mouse hearts and used multiplexed, quantitative proteomics 6,7 to track hundreds of proteins in proximity of Ca V 1.2. We observed that the Ca 2+ channel inhibitor Rad 8,9 , a monomeric G-protein, is enriched in the Ca V 1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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