Key points Prevailing dogma holds that activation of the β‐adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L‐type Ca V 1.2 channel activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca V 1.2 channel clusters decorate T‐tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca 2+ ‐dependent co‐operative gating behaviour mediated by physical interactions between adjacent channel C‐terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A‐dependent augmentation of Ca V 1.2 channel abundance along cardiomyocyte T‐tubules, resulting in the appearance of channel ‘super‐clusters’, and enhanced channel co‐operativity that amplifies Ca 2+ influx. On the basis of these data, we suggest a new model in which a sub‐sarcolemmal pool of pre‐synthesized Ca V 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation–contraction coupling. Abstract Voltage‐dependent L‐type Ca V 1.2 channels play an indispensable role in cardiac excitation–contraction coupling. Activation of the β‐adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca V 1.2 activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. Ca V 1.2 channels exhibit a clustered distribution along the T‐tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca 2+ ‐dependent co‐operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C‐terminal tails. This amplifies Ca 2+ influx and augments myocyte Ca 2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter Ca V 1.2 channel clustering to facilitate co‐operative channel interactions and elevate Ca 2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca V 1.2–Ca V 1.2 physical interactions. Super‐resolution nanoscopy and dynamic channel tracking indicate tha...

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large “superclusters” wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered “emptying” of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

Efficient access to C(10) of (À)-cytisine via C-H activation provides access to enantiomerically pure nicotinic acetylcholine receptor ligands that target the highaffinity nicotine a4b2 subtype with enhanced selectivity. These C(10) cytisine variants retain a partial agonist profile at the a4b2 subtype but, critically, display negligible activity at the a7 receptor subtype. Using computational methods, Gallagher and colleagues link receptor selectivity to key protein residues associated with, as well as beyond, the immediate ligand binding site.

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The insect GABA receptor, RDL (resistance to dieldrin), is a cys-loop ligand-gated ion channel (cysLGIC) that plays a central role in neuronal signaling, and is the target of several classes of insecticides. Many insects studied to date possess one Rdl gene; however, there is evidence of two Rdls in aphids. To characterise further this insecticide target from pests that cause millions of dollars’ worth of crop damage each year, we identified the complete cysLGIC gene superfamily of the pea aphid, Acyrthosiphon pisum, using BLAST analysis. This confirmed the presence of two Rdl-like genes (RDL1 and RDL2) that likely arose from a recent gene duplication. When expressed individually in Xenopus laevis oocytes, both subunits formed functional ion channels gated by GABA. Alternative splicing of RDL1 influenced the potency of GABA, and the potency of fipronil was different on the RDL1bd splice variant and RDL2. Imidacloprid and clothianidin showed no antagonistic activity on RDL1, whilst 100 μM thiacloprid reduced the GABA responses of RDL1 and RDL2 to 55% and 62%, respectively. It was concluded that gene duplication of Rdl may have conferred increased tolerance to natural insecticides, and played a role in the evolution of insect cysLGICs.