MUSCARINIC RECEPTOR IS COUPLED WITH A CATION CHANNEL THROUGH A GTP-BINDING PROTEIN IN GUINEA-PIG CHROMAFFIN CELLS

1. The ionic current evoked by muscarinic receptor agonists was investigated in dispersed chromaffin cells of the guinea-pig adrenal medulla using the whole-cell version of the patch-clamp procedure. 2. Muscarine or oxotremorine (0.03-10-mu-M) produced an inward current associated with an increase in current noise at a holding potential of -40 mV. The relationship between current and oxotremorine concentration fitted well to a rectangular hyperbole with an apparent dissociation constant (K(A)) of 0.23-mu-M. 3. The muscarinic antagonists pirenzepine (0.1-mu-M) and AF-DX 116 (0.3-mu-M) shifted the dose-response curve to the right in a parallel manner. Dissociation constants (K(B)) for pirenzepine and AF-DX 116 were estimated to be 50 and 70 nM, respectively. 4. The current-voltage relation for the current induced by muscarine had a negative slope below -30 or -20 mV, and the current reversed its polarity at 0.4 +/- 0.8 mV (n = 4) in standard salt solution. Removal of Mg2+ or Ca2+ from the perfusate did not modify the muscarinic current-voltage relationship. 5. When Na+ in the bath solution was replaced with Tris, the muscarinic current-voltage relationship shifted to the left (the hyperpolarizing direction); the current reversed its polarity at -18.7 +/- 1.2 mV in a solution containing 72 mM-Na+ (three cells) and at -57.5 +/- 2.7 mV in nominally Na+-free solution (three cells). When Ca2+ was replaced by Mg2+, in Na+-free solution, an inward current was not evoked by muscarinic stimulation. 6. Tetraethylammonium (TEA; 0.03-3 mM) reduced the muscarinic current at -40 mV, and the K(D) value was 0.34 mM with a Hill coefficient of 1. Barium (4 mM) reduced the current to 0.69 +/- 0.06 of control (n = 3). 7. When the recording electrodes contained guanosine-5'-O-(3-thiophosphate) (GTP-gamma-S, 100-mu-M), an inward current developed at -55 mV during the first few minutes after breaking into the cell interior. This inward current was associated with an increase in noise and was not larger at -70 mV than at -55 mV; it reached a peak value about 3-4 min after breaking into the cell interior and then declined over the next 2-3 min. 8. Muscarinic agonists had no effect in those cells in which intracellular GTP-gamma-S had first evoked a transient inward current. The inward current caused by nicotine was unaffected by intracellular GTP-gamma-S. These results indicate that intracellular GTP-gamma-S transiently activates, and then inactivates, the muscarinic cation channel. 9. Following pre-treatment with pertussis toxin (PTX), the muscarinic current was almost abolished and the GTP-gamma-S-induced inward current was significantly inhibited. Nicotinic currents were unaffected. 10. It is concluded that activation of the muscarinic receptor in chromaffin cells increases the cation conductance to Na+, K+ and Ca2+ through activation of a PTX-sensitive GTP-binding protein and that the receptor involved has high affinities for pirenzepine and AF-DX 116.