To investigate possible effects of adrenergic stimulation about G proteinCactivated inwardly rectifying K+ channels (GIRK), acetylcholine (ACh)-evoked K+ current, IKACh, was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the 2-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the -adrenergic receptor (2AR) by PKA was excluded by using a mutant 2AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the subunit of G proteins (Gs) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of indicated Gs, GIRK currents were inhibited, presumably due to sequestration of the / subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also controlled by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in oocytes, -adrenergic activation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from 2AR. oocytes inside a membrane-delimited manner ( Schreibmayer et al. 1996). This increases the possibility that such inhibition may occur in atrial myocytes when sympathetically stimulated. The current study investigates the effects of -adrenergic activation on GIRK channels in native rat atrial cells and in the oocyte manifestation system. We find that, in atrial myocytes as well as with oocytes, the main effect of -adrenergic activation is an enhancement of the ACh-induced GIRK current via PKA-catalyzed phosphorylation. The prospective for this phosphorylation is still unclear, but it is definitely downstream from your -adrenergic receptor. MATERIALS AND METHODS Electrophysiology Atrial cells were enzymatically disaggregated Mitoxantrone enzyme inhibitor from hearts of adult Sprague-Dawley rats as explained (Dascal et al. 1993) and stored in the incubator for 1C3 d at 37C in M199 under 95% Mitoxantrone enzyme inhibitor O 2/5% CO2 until electrophysiological recordings were performed. Cells were then pipetted into a plastic petri dish having a diameter of 35 mm, mounted to a Peltier element thermostated object Mitoxantrone enzyme inhibitor holder of an inverted microscope (IM35; Carl Zeiss, Inc.). Patch-clamp current recordings were performed with an Axopatch 1D amplifier (Axon Devices) using fire-polished pipettes with an open tip resistance of 2C4 M, drawn from standard hematocrit capillaries (564, L/M-3P-A puller; List Electronik). Whole cell current recordings were acquired by rupturing the membrane patch under the tip of the Nafarelin Acetate glass pipette by suction. Pipette solutions for perforated patch recordings were made by combining 100 l of a nystatin stock answer (50 mg/ml nystatin dissolved in DMSO in the original answer) with 10 ml pipette answer, sonicated and filtered through 200-m ultrafilters before use. The amount of nystatin needed to get electrical perforation within 1C2 min after gigaseal formation assorted within a range of 1C5 and depended within the atrial cell preparation. Current recordings were acquired by keeping the cell’s membrane potential constant at ?80 mV and superfusing with HP medium (see below) containing 10?5 M acetylcholine and/or 10?6 M isoproterenol. H89 oocytes were prepared as explained (Dascal and Lotan 1992), and the following amounts of cRNA were injected (ng/oocyte): 0.1C1.5 muscarinic m2-receptor (m2R), 0.05C0.75 wild-type -adrenergic receptor (2ARwt), 0.05C0.75 2ARPF, 0.0025C0.2 GIRK1 wt, 0.0025C0.0075 GIRK1C40, 0.0125C0.2 GIRK4wt, 0.0125C0.0375 GIRK4 C40, 0.075C0.75 GIRK2wt, 0.02C2 Gs, 1.5 G2, 1.5 G1, 10 CFTR. To knock out the endogenously existing GIRK5 subunit, 80 ng/oocyte of the phosphothioated antisense oligonucleotide KHA2 was injected together with the cRNAs (Hedin et al. 1996). Oocytes were kept in NDE at 19C21C in an incubator for 3C10 d before electrophysiological recordings. Oocytes were placed in a recording chamber that allowed superfusion with extracellular medium by gravity circulation at 19C21C and currents were recorded with the two-electrode voltage clamp.