Abstract
Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.
Highlights
We investigated whether A1 receptors (A1R) exhibits a voltage-dependent interaction with its physiological agonist Ado, by measuring IKACh activation in guinea pig cardiomyocytes
We analyzed the effect of voltage on the deactivation kinetics of IKACh, as this reflects the rate of agonist unbinding, and is a comparative index of affinity [28,29,30]
Despite M2 receptors (M2R) and A1R activate the same downstream signaling pathway in cardiac myocytes, in this work we found a differential voltage-dependent modulation of IKACh by the physiological agonists ACh and Ado, that is, a null or very weak influence of voltage on the IKACh evoked by Ado-A1R that made this current to display no relaxation behavior
Summary
Stimulation of both cholinergic muscarinic M2 receptors (M2R) and adenosine A1 receptors (A1R) has an important physiological impact on the electrophysiology and mechanical. In cat atrial myocytes we have shown that M2R exhibits agonist-specific voltage dependence, where the intrinsic voltage sensitivity of this receptor [7,8,9] modifies its affinity for diverse agonists in a ligand-selective manner, which is eventually reflected on the activation of the coupled KACh channels [10]. This property can be distinguished in the deactivation kinetics of the current carried by these channels, IKACh [11]. In this work we used guinea-pig atrial myocytes (where this pathway is operative [25, 26]) to assess the effects of membrane potential on the IKACh activation and the ability to induce a voltage-dependent hallmark of the current (relaxation) when the signaling pathway is triggered by adenosine (Ado) through A1R
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