1. Whole‐cell patch‐clamp technique was used to study the beta‐adrenergic and cholinergic regulation of the inwardly rectifying K+ conductance (gK1) in isolated guinea‐pig ventricular myocytes. 2. In Cl(‐)‐free solutions or in the presence of 9‐anthracenecarboxylic acid or Co2+, bath‐applied isoprenaline (Iso) partially inhibited the steady‐state whole‐cell conductance (gss) calculated from the steady‐state current (Iss)‐voltage (Iss‐V) curve at membrane voltages (Vm) negative to the equilibrium potential for potassium (EK). Iss was also inhibited at Vm positive to EK when the extracellular [K+] was 20 mM. The Iso‐sensitive component of gss exhibited the characteristics of the inwardly rectifying K+ conductance (gK1). 3. The Iso‐induced inhibition of gK1 was reversible, concentration dependent, blocked by propranolol, mimicked by both forskolin and dibutyryl cAMP, and prevented by including a cAMP‐dependent protein kinase (PKA) inhibitor in the pipette solution. These findings suggest that PKA mediates the Iso‐induced inhibition of gK1. 4. The apparent dissociation constant (KD) for the concentration dependence of Iso‐induced inhibition was 0.035 microM and the Hill coefficient was approximately 1.0. A maximal Iso concentration (1 microM) inhibited gK1 by 40 +/‐ 4.1% (mean +/‐ S.E.M.; n = 13). 5. Bath application of acetylcholine (ACh, 0.1 microM or more) antagonized the Iso‐induced (1 microM) inhibition of gK1; [ACh] > 1.0 microM antagonized 88 +/‐ 2.1% (n = 10) of the inhibition. ACh increased the KD for Iso to inhibit Iso‐sensitive gK1 and also reduced the maximal Iso‐induced inhibition. 6. ACh‐induced antagonism could be abolished by pre‐incubating myocytes with pertussis toxin (PTX), suggesting that a muscarinic receptor‐coupled, PTX‐sensitive G protein, Gi, is involved. 7. ACh (10 microM) also antagonized approximately 70% of the dibutyryl cyclic AMP (1 mM)‐induced inhibition of gK1 (n = 3), suggesting that the ACh‐induced antagonism involves more than simply inhibiting the Iso‐mediated activation of adenylyl cyclase via the activated Gi. 8. Intracellularly applied okadaic acid (OkA, 1 microM) did not alter gK1 (control = 134 +/‐ 5.1 nS vs. OkA = 136 +/‐ 6.1 nS), but the Iso‐induced decrease in gK1 was less (P < 0.001) with OkA present (42.1 +/‐ 2.4 nS, n = 5) than when absent (54.0 +/‐ 2.2 nS, n = 10). However, ACh (10 microM) failed to antagonize Iso‐induced inhibition with OkA present, suggesting involvement of a protein phosphatase.
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