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Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

ABSTRACT: BACKGROUND AND PURPOSE:The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca(2+) currents carried by the Cav 1.2 channels [ICa1.2 ] of rat cardiomyocytes. As only few drugs stimulate ICa1.2 , this study was designed to analyse the effects of MuA on vascular Cav 1.2 channels. EXPERIMENTAL APPROACH:Vascular activity was assessed on rat aorta rings mounted in organ baths. Cav 1.2 Ba(2+) current [IBa1.2 ] was recorded in single rat aorta and tail artery myocytes by the patch-clamp technique. Docking at a 3D model of the rat, ?1c central pore subunit of the Cav 1.2 channel was simulated in silico. KEY RESULTS:In rat aorta rings MuA, at concentrations ?14.2 ?M, increased 30 mM K(+) -induced tone and shifted the concentration-response curve to K(+) to the left. Conversely, at concentrations >14.2 ?M, it relaxed high K(+) depolarized rings and antagonized Bay K 8644-induced contraction. In single myocytes, MuA stimulated IBa1.2 in a concentration-dependent, bell-shaped manner; stimulation was stable, incompletely reversible upon drug washout and accompanied by a leftward shift of the voltage-dependent activation curve. MuA docked at the ?1C subunit central pore differently from nifedipine and Bay K 8644, although apparently interacting with the same amino acids of the pocket. Neither Bay K 8644-induced stimulation nor nifedipine-induced block of IBa1.2 was modified by MuA. CONCLUSIONS AND IMPLICATIONS:Murrayafoline A is a naturally occurring vasoactive agent able to modulate Cav 1.2 channels and dock at the ?1C subunit central pore in a manner that differed from that of dihydropyridines.

SUBMITTER: Saponara S

PROVIDER: S-EPMC5341225 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

Saponara S S Durante M M Spiga O O Mugnai P P Sgaragli G G Huong T T TT Khanh P N PN Son N T NT Cuong N M NM Fusi F F

British journal of pharmacology 20151223 2

<h4>Background and purpose</h4>The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca(2+) currents carried by the Cav 1.2 channels [ICa1.2 ] of rat cardiomyocytes. As only few drugs stimulate ICa1.2 , this study was designed to analyse the effects of MuA on vascular Cav 1.2 channels.<h4>Experimental approach</h4>Vascular activity was assessed on rat aorta rings mounted in organ baths. Cav 1.2 Ba(2+) current [IBa1.2 ] was recorded in single rat aorta and tail artery myocyt ...[more]

PMID: 26493241

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Project description:KEY POINTS:Prevailing dogma holds that activation of the ?-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. ABSTRACT:Voltage-dependent L-type CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the ?-adrenergic receptor (?AR)/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 ?AR 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 ?AR 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 ?AR-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

Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

Publications

Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

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