Electrophysiological characterization of cloned m1 muscarinic receptors expressed in A9 L cells

Abstract

The electrophysiological properties of A9 L cells stably transfected with m1 muscarinic receptor cDNA were examined by using the whole-cell patch-clamp technique. In current-clamp recordings, acetylcholine (AcCho) elicited a hyperpolarization of all transfected cells studied but had no effect on nontransfected A9 L cells. In voltage-clamp recordings, AcCho elicited an outward current at -50 mV accompanied by an increase in conductance. The onset of the current response was consistently delayed by several seconds with respect to the onset of the application of AcCho and could not be accounted for by diffusion. The AcCho-induced currents were reversibly inhibited by the muscarinic receptor antagonist atropine (1 microM) but were unaffected by the nicotinic receptor antagonist tubocurarine (50 microM). Ion-substitution experiments replacing K+ with N-methyl-D-glucamine and Cl- with methanesulfonate indicated that the current was carried mainly by K+, although a minor part appeared to be carried by Cl-. The AcCho-induced current could be blocked by the K+ channel blocking agents tetraethylammonium ion, 4-aminopyridine, apamin, and Ba2+ but not by Cs+. The AcCho-induced current was inhibited when 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was included in the patch pipette or when extracellular Cd2+ or Co2+ was applied, indicating a role for intracellular Ca2+ in the generation of the response. Thus, these results show that cloned m1 muscarinic receptors expressed in A9 L cells can activate a Ca2+-dependent K+ conductance, possibly via a second-messenger system.