Dose-response of acetylcholine receptor channels opened by a flash-activated agonist in voltage-clamped rat myoballs

Abstract

Whole‐cell or single‐channel currents through acetylcholine (ACh) receptor channels were studied in voltage‐clamped rat myoballs or in excised membrane patches from myoballs. The recording pipette contained CsCl to suppress outward currents, and tetrodotoxin was used to help suppress Na+ currents. To minimize problems associated with bath applied agonists, myoballs were bathed in a solution containing the inactive (cis) isomer of the photo‐isomerizable azobenzene derivative, Bis‐Q. Calibrated light flashes of varying intensity were presented to produce concentration jumps of agonist, trans‐Bis‐Q. The resulting whole‐cell current relaxations through ACh channels approach a steady state along an exponential time course, then decline as the newly created agonist diffuses away over the next few seconds. The dose‐response relationship was inferred from Hill (double‐log) plots for myoballs bathed in 500 nM‐cis‐Bis‐Q at three membrane potentials. At low agonist concentrations (less than 300 nM‐trans‐Bis‐Q), the slope of the Hill plot averaged 1.62 at ‐150 mV, 1.89 at ‐100 mV, and 2.05 at +80 mV. These results are consistent with an apparent agonist affinity constant that decreases with membrane depolarization and shifts the responses further down on the dose‐response curve. When the myoballs were bathed in higher concentrations of cis‐Bis‐Q (1.5‐20 microM), the slope of the Hill plot was reduced at all membrane potentials, although it was still closer to two at positive potentials. This is expected from the known sigmoid shape of the dose‐response relation. The shallow dependence of the Hill slope on agonist concentration suggests the presence of negative cooperativity in the over‐all binding of agonist molecules. Following treatment of the membrane with dithiothreitol to reduce disulphide groups, the Hill slope for the reversibly bound agonist, trans‐Bis‐Q, remained near two. The kinetics of currents at hyperpolarized membrane potentials became complicated at higher agonist concentrations in a manner that was consistent with open‐channel block by trans‐Bis‐Q; the currents showed a slow secondary increase in conductance. Averaged single‐channel recordings at higher agonist concentrations resemble macroscopic relaxations under comparable conditions. Furthermore, those recordings also suggested that open channels are blocked by trans‐Bis‐Q at concentrations greater than 2 microM; the block depends strongly on membrane potential and increases with hyperpolarization. Currents at positive membrane potentials showed no evidence of open‐channel block.

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