Activation and Ca2+ permeation of stably transfected alpha 3/beta 4 neuronal nicotinic acetylcholine receptor

The alpha 3/beta 4 rat neuronal nicotinic acetylcholine receptor, stably tr ansfected in human embryonic kidney cells, was examined using the whole-cel l-clamp technique and 2-dimensional confocal imaging. Application of agonis ts (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwa rdly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 va lues of congruent to 22 and congruent to 64 mu M, respectively, and suggest ed second order binding kinetics (Hill coefficient similar to 2). The time constant of desensitization (decay) of nicotine;activated current was conce ntration-dependent (typically similar to 10 s at 30 mu M versus similar to 1.0 s at 100-1000 mu M), but not voltage-dependent and was significantly sm aller than the similar to 200 s reported for the alpha 3/beta 4 receptor ex pressed in Xenopus oocytes. Nicotine-activated current was rapidly and reve rsibly blocked by coapplication of mecamylamine and d-tubocurarine. At -80 mV holding potentials, the current was also suppressed by similar to 25% ei ther upon complete removal or elevation of Ca2+ to 10 mM. Total replacement of Na+ by Ca2+ also completely blocked the current. On the other hand, evi dence for permeation of Ca2+ was indicated by increased inward current at - 40 mV upon elevation of Ca2+ from 2 to 10 mM, as well as a vise in the cyto solic Ca2+ proportional to the current carried by the receptor. These findi ngs are consistent with the idea that Ca-,(2+) in addition to its channel-p ermeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha 3/beta 4 neuronal nicotinic acetyl choline receptor, when stably expressed in human embryonic kidney 293 cells , has desensitization kinetics and Ca2+ regulatory mechanisms somewhat diff erent from those described for the receptor expressed in Xenopus oocytes.