Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist.

The patch-clamp technique was used to examine the activation of single acetylcholine receptor channels of clonal BC3H-1 mouse muscle cells. Single-channel currents were activated by low concentrations of the strong agonists acetylcholine (ACh, 50-100 nM), carbamylcholine (1-2 microM), and suberyldicholine (30-50 nM). At low agonist concentrations channel openings occur as isolated short-duration openings and as bursts of longer duration openings separated by brief closed periods. Two distinct types of brief closed periods separate long duration openings: brief closures (mean duration, 50 microseconds) and intermediate closures (mean duration, 0.5-1.0 ms). The kinetic properties of intermediate closures depend on the agonist, suggesting that they reflect receptor reopening from the closed state leading to the open state. Properties of brief closures, in contrast, are independent of the agonist, indicating that they result from an additional closed state leading away from the pathway producing the open state. A receptor activation scheme is proposed which accounts for the observed closed states, and transition rate estimates are presented for steps within the proposed scheme. The channel opening rate, beta, differs several-fold for the agonists studied (200-1400 s-1) and is comparable to the dissociation rate, k-2 (900 s-1). The dissociation rate is similar for the three agonists studied. The channel closing rate, alpha, is much slower than the opening rate (20-60 s-1). The probability is high that a doubly liganded channel is in the open state and depends on the agonist (0.75-0.97). Beta increases and alpha decreases at more negative membrane potentials, whereas k-2 shows little potential dependence.