The voltage-sensitive cardiac M2 muscarinic receptor modulates the inward rectification of the G protein-coupled, ACh-gated K+ current

Abstract

The acetylcholine (ACh)-gated inwardly rectifying K current (I) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of I. Here, we investigated the relative contribution of two distinct mechanisms of I inward rectification measured in atrial myocytes: a rapid component due to K channel block by intracellular Mg and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when I was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (MR) and directly stimulates trimeric G proteins to open K channels. Moreover, the time-dependent component of I inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, MR; consistent with our previous work demonstrating that voltage-dependent conformational changes in the MR alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of I indicate that rapid Mg-polyamine block accounts for 60-70% of inward rectification, with MR voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the K channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.

Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine

“…As a molecular mechanism, our findings are not consistent with the concept that M 2 R voltage dependence arises from voltage-induced transitions between the high-and low-affinity states of the receptor when coupled or not to its cognate G-protein, respectively [8,28,35,36], since this view is contradictory with evidence showing diverse voltage-dependent effects of different agonists on the same GPCR [11,15,16,21,22,32,33]. Alternatively, our data are more compatible with the idea that membrane potential induces conformational changes directly at the agonist binding (orthosteric) site of GPCRs, independent of G-protein coupling, which determine the modulation of the remainder signaling pathway [9,11,15,20,22].…”

“…In previous studies, we described the agonist-specific voltage sensitivity of M 2 R, where membrane depolarization reduces, augments, or not modifies the receptor activation by several muscarinic agonists [9,11,16]. Here, we investigated whether A 1 R exhibits a voltage-dependent interaction with its physiological agonist Ado, by measuring ).…”

“…Also, we previously proposed that this is the molecular mechanism underlying a very distinctive attribute of receptor-stimulated Kir3.x currents (including I KACh ), known as relaxation [12], which consists of a time-dependent augment or reduction of the current upon hyperpolarizing or depolarizing, respectively, the cardiomyocyte membrane with voltage steps [13,14]. Agonists with higher affinity at hyperpolarized potentials induce I KACh to manifest its typical relaxation behavior, whereas those ligands with inverse voltage dependence, i.e., higher affinity at depolarized voltages, lead to this current to display the appearance of an "opposite" relaxation, with delayed rectifying characteristics [12,15,16].…”

Computational Insights Into Voltage Dependence of Polyamine Block in a Strong Inwardly Rectifying K+ Channel

“…A clear limitation of our study is the fact that so far we tested only two polyamines PUT 2+ and SPM 4+ on an "atypical" strong inward rectifier channel that has been poorly studied in the homotetrameric form. Studies investigating polyamine block in Kir3 channels have almost exclusively focused on the heterotetrameric versions composed of Kir3.1 and Kir3.4 (carrying I KACh currents) (Yamada and Kurachi, 1995;Salazar-Fajardo et al, 2018). However, strong inward rectification was confirmed for the crystal structure construct of Kir3.2 (Whorton and MacKinnon, 2011), justifying the use of this construct to study the mechanisms of inward rectification.…”

Cyclic Stretch Promotes Proliferation and Contraction of Human Bladder Smooth Muscle Cells by Cajal-Mediated c-kit Expression in Interstitial Cells

“…The bladder detrusor muscle simultaneously expresses M2 and M3 subtypes of M receptors, but M3 is the only one directly related to detrusor contraction [15,16]. M2 synergizes with beta receptors to inhibit detrusor relaxation and assist in urination under the action of large doses of Ach [17,18].…”