Activation and Ca2+ permeation of stably transfected alpha3/beta4 neuronal nicotinic acetylcholine receptor

The alpha3/beta4 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 with22 and congruent with64 microM, respectively, and suggested second order binding kinetics (Hill coefficient approximately 2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically approximately 10 s at 30 microM versus approximately 1.0 s at 100-1000 microM), but not voltage-dependent and was significantly smaller than the approximately 200 s reported for the alpha3/beta4 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 approximately 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 rise in the cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca2+, in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha3/beta4 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.