Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors

A new approach was developed to determine quantitatively the fraction of current carried by Ca²⁺ through an ion channel under physiological conditions. This approach entails the simultaneous measurement of membrane current and intracellular Ca²⁺ for single cells. Whole-cell patch-clamp techniques were used to measure current, and intracellular Ca²⁺ was monitored with the fluorescent indicator fura-2. To obtain a quantitative measure of the fraction of current carried by Ca²⁺, a cell-by-cell calibration method was devised to account for differences among cells in such factors as cellular volume and Ca²⁺ buffering. The method was used to evaluate the Ca²⁺ flux through muscle and neuronal nicotinic ACh receptors (nAChRs). In a solution containing 2.5 mM Ca²⁺ at a holding potential of - 50 mV, Ca²⁺ carries 2.0% of the inward current through muscle nAChRs from BC3H1 cells and 4.1% of the inward current through neuronal nAChRs from adrenal chromaffin cells. The Ca²⁺ flux through neuronal nAChRs of adrenal chromaffin cells is insensitive to α-bungarotoxin. The influx of Ca²⁺ is voltage dependent, and because of the Ca²⁺ concentration difference across the cellular membrane, there is Ca²⁺ influx into the cell even when there is a large net outward current. At both muscle and neuronal cholinergic synapses, activity-dependent Ca²⁺ influx through nicotinic receptors produces intracellular signals that may have important roles in synaptic development, maintenance, and plasticity.