Activation and Ca2+ permeation of stably transfected α3/β4 neuronal nicotinic acetylcholine receptor

The alpha 3/beta 4 rat neuronal nicotinic acetylcholine receptor, stably transfected in human embryonic kidney cells, was examined using the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 values of congruent to 22 and congruent to 64 mu M, respectively, and suggested second order binding kinetics (Hill coefficient similar to 2). The time constant of desensitization (decay) of nicotine;activated current was concentration-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 smaller than the similar to 200 s reported for the alpha 3/beta 4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine and d-tubocurarine. At -80 mV holding potentials, the current was also suppressed by similar to 25% either 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, evidence 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 cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca-,(2+) in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha 3/beta 4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different from those described for the receptor expressed in Xenopus oocytes.