Abstract

The α3/β4 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 ≅22 and ≅64 μM, respectively, and suggested second order binding kinetics (Hill coefficient ∼2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically ∼10 s at 30 μM versus ∼1.0 s at 100–1000 μM), but not voltage-dependent and was significantly smaller than the ∼200 s reported for the α3/β4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine andd-tubocurarine. At −80 mV holding potentials, the current was also suppressed by ∼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 α3/β4 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 inXenopus oocytes.