HUMAN ALPHA-4-BETA-2 NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTOR IN HEK-293 CELLS - A PATCH-CLAMP STUDY

The cloning and expression of genes encoding for the human neuronal ni cotinic acetylcholine receptors (nAChRs) has ii opened new possibiliti es for investigating their physiological and pharmacological propertie s. Cells (HEK 293) stably transfected with two of the major brain subu nits, alpha 4 and beta 2, were characterized electrophysiologically us ing the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC(50 )) of 3 mu M and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human alpha 4 beta 2 receptors is (- )-nicotine > ACh > (-)-cytisine > ABT-418. At saturating concentration s, the efficacy of these ligands is ABT-418 much greater than (-)-nico tine > ACh much greater than (-)-cytisine > GTS-21 (previously named D MXB). Coapplication of 1 mu M ACh with known nAChR inhibitors such as dihydro-beta-erythroidine and methyllycaconitine reversibly reduces th e current evoked by the agonist with respective IC50 values of 80 nM a nd 1.5 mu M. The current-voltage relationship of human alpha 4 beta 2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human alpha 4 beta 2 nAChRs are perm eable to sodium and potassium ions. In the ''outside-out'' configurati on, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is ob served when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.