RESUMO
The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) 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 IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh 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, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh 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.
Assuntos
Potenciais de Ação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Miócitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animais , Gatos , Células Cultivadas , Feminino , Átrios do Coração/citologia , Magnésio/metabolismo , Masculino , Miócitos Cardíacos/fisiologia , Poliaminas/metabolismoRESUMO
The human intestinal pathogen Giardia lamblia is a flagellated unicellular protozoan with pronounced medical and biological relevance. However, the basic physiology of Giardia trophozoites has been sparsely studied, especially the electrical and ionic properties of their cellular membrane which are virtually unknown. In this study, we were able to record and characterize the macroscopic ionic currents of Giardia trophozoite membrane by electrophysiological methods of the patch clamp technique. Giardia trophozoites showed a high current density (â¼600 pA/pF at -140 mV) that was activated upon hyperpolarization. This current was carried by a chloride-selective channel (I Cl-G) and it was the most important determinant of the membrane potential in Giardia trophozoites. Moreover, this conductance was able to carry other halide anions and the sequence of permeability was Br(-) > Cl(-) ≈ I(-) â« F(-). Besides the voltage-dependent inward-rectifying nature of I Cl-G, its activation and deactivation kinetics were comparable to those observed in ClC-2 channels. Extracellular pH modified the voltage-dependent properties of I Cl-G, shifting the activation curve from a V 1/2 = -79 ± 1 mV (pH 7.4) to -93 ± 2 mV (pH 8.4) and -112 ± 2 mV (pH 5.4). Furthermore, the maximal amplitude of I Cl-G measured at -100 mV showed dependence to external pH in a bell-shaped fashion reported only for ClC-2 channels. Therefore, our results suggest that I Cl-G possesses several functional properties similar to the mammalian ClC-2 channels.