Concentration-dependent effects of intracellular GTP on activation of the muscarinic K + channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.i ~I ACh. GTP (0.01-30 ~M) and 0.5 mM MgCI 2 were applied to the inside side of the patch membrane. K + channels were activated with GTP concentration above 0.i ~M. Channel activation reached a maximal value with 1-3 DM GTP. It decreased at GTP concentrations larger than 3 LM, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K + channel may play an important role in amplifying the signal from the membrane receptor to the K + channel. Key Words: muscarinic K + channel, intracellular GTP, positive cooperativity 136.5, KCI 5.4, CaCI 2 1.8, MgCI 2 0.53, glucose 5.5, HEPES-NaOH 5 (pH 7.4). In the "inside-out" patch experiments, the bath was superfused with artificial internal solution (in mM): KCI 140, MgCI 2 0.5, EGTA 5, HEPES-KOH 5 (pH 7.2). GTP (Na salt, Sigma) was added to the internal solution in various concentrations. All experiments were performed at 35-37~
The channel activity was measured after having reached a steady state at each concentration of GTP and was expressed in terms of N'Po, where N is the number of the K + channel in the patch and Po is the open probability of each channel. The relative N-Po at various concentrations of GTP was obtained with reference to the value at 1 ~ GTP in each patch. The relation between relative NoPo and GTP was fitted by equation (i), using the nonlinear least squares regression method MULTI (Yamaoka et al., 1981).