DIRECT G PROTEIN GATING OF ACH-SENSITIVE K⁺ CHANNEL IN GUINEA-PIG ATRIAL CARDIOMYOCYTES : ANALYSIS BY INSIDE-OUT PATCH CLAMP MODE

Abstract

Direct actions of guanosine 5'-triphosphate (GTP)-binding protein (G protein) on the K⁺ channel in isolated single atrial muscle cells from guinea-pig hearts were investigated. In cell-attached patch-clamp mode, ACh-sensitive K⁺ (K⁺ [ACh]) channels were stimulated by application of carbachol (10 μM). A unitary channel current identified by carbachol responsiveness was activated inwardly at the resting membrane potential, and was decreased with an increase in membrane potential. At 0 mV, the single-current was zero mV (the reversal potential). The extracellular and pipette solutions (140 mM KCl) were similar. It was a slope conductance of 44.7±3.5 pS (n=8) and an average open time at -80 mV of 1.4±0.3 msec (n=7). It had a strong inwardly-rectifier property at positive potentials. The response was blocked by the addition of atropine (1 μM) to the pipette solution. On switching from a cell-attached to an inside-out patch-clamp experiment (to avoid the influences of intracellular signal transductions), the activation of K⁺ [ACh] channels was gradually attenuated. Holding potential was -80 mV. But since other channels (i. e. ATP-sensitive K⁺ channel and inward-rectifying K^+ channel) still remained, the cell was transiently taken out from the bath solution (exposed to the air) to inactivate the channels. GTP (100 μM), which was applied by a concentration-clamp technique, activated the K⁺ [ACh] channels with the same characteristics within a moment of its application. The responses were reversible. These results indicate that G protein directly activates the K⁺ [ACh] channels on the atrial muscle cell membrane, independent of second messengers.