The cardiac muscarinic receptor (M2R) regulates heart rate, in part, by modulating the acetylcholine (ACh) activated K+ current IK,ACh through dissociation of G-proteins, that in turn activate KACh channels. Recently, M2Rs were noted to exhibit intrinsic voltage sensitivity, i.e. their affinity for ligands varies in a voltage dependent manner. The voltage sensitivity of M2R implies that the affinity for ACh (and thus the ACh effect) varies throughout the time course of a cardiac electrical cycle. The aim of this study was to investigate the contribution of M2R voltage sensitivity to the rate and shape of the human sinus node action potentials in physiological and pathophysiological conditions. We developed a Markovian model of the IK,ACh modulation by voltage and integrated it into a computational model of human sinus node. We performed simulations with the integrated model varying ACh concentration and voltage sensitivity. Low ACh exerted a larger effect on IK,ACh at hyperpolarized versus depolarized membrane voltages. This led to a slowing of the pacemaker rate due to an attenuated slope of phase 4 depolarization with only marginal effect on action potential duration and amplitude. We also simulated the theoretical effects of genetic variants that alter the voltage sensitivity of M2R. Modest negative shifts in voltage sensitivity, predicted to increase the affinity of the receptor for ACh, slowed the rate of phase 4 depolarization and slowed heart rate, while modest positive shifts increased heart rate. These simulations support our hypothesis that altered M2R voltage sensitivity contributes to disease and provide a novel mechanistic foundation to study clinical disorders such as atrial fibrillation and inappropriate sinus tachycardia.
While chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K+ current IK1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac IK1. The voltage- and K+-dependence of chloroquine block implied that the binding site was located within the ion conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine binding site within the cytoplasmic pore domain, rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion channel blockers, chloroquine does not bind within the transmembrane pore. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block IK1 even in the presence of high affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1 blocking properties.