Dataset Information

Adrenergic Ca_V1.2 Activation via Rad Phosphorylation Converges at α_1C I-II Loop.

ABSTRACT:

Rationale

Changing activity of cardiac Ca_V1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac Ca_V1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic.

Objective

The primary goal of this study was to determine how various factors converging at the Ca_V1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure.

Methods and results

We generated transgenic mice with expression of Ca_V1.2 α_1C subunits with (1) mutations ablating interaction between α_1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α_1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α_1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of Ca_Vβ to α_1C I-II loop and eliminated β-adrenergic agonist stimulation of Ca_V1.2 current. In contrast, introduction of the exon 9* splice variant in the α_1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β_2B and Rad or transgenically expressed in cardiomyocytes.

Conclusions

Ca²⁺ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α_1C I-II loop. Ca_Vβ binding to α_1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca²⁺ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α_1C.

SUBMITTER: Papa A

PROVIDER: S-EPMC7790865 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Key pointsPrevailing dogma holds that activation of the β-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -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 CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 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.AbstractVoltage-dependent L-type CaV 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 CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.

Dataset Information

Adrenergic Ca_V1.2 Activation via Rad Phosphorylation Converges at α_1C I-II Loop.

Rationale

Objective

Methods and results

Conclusions

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Tweets

Dataset Information

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

Rationale

Objective

Methods and results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Adrenergic Ca_V1.2 Activation via Rad Phosphorylation Converges at α_1C I-II Loop.