On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

Abstract

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K ÷ channel (IKaeh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-0-(3-thiotriphosphate) (GTP-,/S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentrationdependent manner in ~80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-3,S. The agonistindependent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IKACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IKACh in a concentrationdependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and G K is essential for basal stimulation oflKach, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

Agonist‐independent effects of muscarinic antagonists on Ca2+ and K+ currents in frog and rat cardiac cells.

“…As will be outlined in the discussion below, our findings are best interpreted by the two following assumptions: (1) an agonist-independent/mAChR-dependent mechanism mediates a spontaneous activation of Gi, as has been recently suggested for GK by Ito, Sugimoto, Kobayashi, Takahashi &Kurachi (1991) andOKabe et al (1991). As a result, cardiac adenylyl cyclase is under a tonic Gi-mediated inhibition which holds back its basal and/or hormonally stimulated activity; (2) in addition to displacing muscarinic agonists from their binding site on the mAChR, atropine and 'M2-selective' antagonists exhibit intrinsic negative activity on the cardiac mAChR which leads to a reduced interaction between the receptor and the corresponding G proteins, Gi and GK.…”

Cellular modelling: experiments and simulation to develop a physiological model of G-protein control of muscarinic K + channels in mammalian atrial cells

“…Differential phosphorylationdependent regulation of the ion channel might underlie this constitutively active K ACh current (Voigt et al 2007). It will be interesting to use our model to investigate possible sources of constitutive K ACh channel activity-activation of K ACh channels by GTP in the absence of ACh (Ito et al 1991) and the loss of Ca 2+ -CaM regulation of RGS proteins might prove to be attractive targets for further investigation. In which context it is interesting to note that knock-out of the RGS4 protein abolished desensitization of the response to ACh in the SA node in mice (Cifelli et al 2008).…”

“…The K ACh currents evoked by various concentrations of ACh were recorded in the whole-cell configuration of the patch-clamp technique. Other experimental data, such as single channel current recording, were taken from published papers (Kurachi et al 1986;Ito et al 1991). …”

INHIBITORY EFFECT OF ^|^beta;-HYDROXYBUTYRIC ACID ON L-TYPE Ca2+ CURRENT UNDER ^|^beta;-ADRENERGIC STIMULATION IN GUINEA PIG CARDIAC VENTRICULAR MYOCYTES

“…Pertussis toxin (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 5 μg was activated by mixing with 50 μl dithiothreitol (100 μM) and 450 μl pipette solution and incubated at 37°C for 15 -20 min. Then, 4.5 ml pipette solution was added, so that the final pertussis toxin (PTX) concentration of in the pipette solution was 1 μg/ml 12,13) . Each experiment was repeated 4 to 5 times.…”