HOW FAST DOES AN ACETYLCHOLINE-RECEPTOR CHANNEL OPEN - LASER-PULSE PHOTOLYSIS OF AN INACTIVE PRECURSOR OF CARBAMOYLCHOLINE IN THE MICROSECOND TIME REGION WITH BC3H1 CELLS

The integrated function of the nervous system depends on specific and rapid transmission of signals between its constituent cells. The nicotinic acetylcholine receptor is the best known of a group of membrane-bound proteins responsible for such transmission; for this process to occur, a specific neurotransmitter, in this case acetylcholine, must bind to the receptor, which then forms transmembrane channels through which cations pass. The resulting change in transmembrane voltage determines whether or not a signal is transmitted. The question of how fast this process takes place in any neurotransmitter receptor has remained one of the interesting and most challenging in the field. To answer it, many attempts have been made to evaluate the rate constant for the opening of the acetylcholine receptor channel, but in almost all these studies the rate was measured after the receptor-mediated reaction, which involves the open channel and many intermediate states, had reached a quasi equilibrium. This resulted in a plethora of reported values for the rate constant that differ by a factor of up to 50-fold, even when the measurements were made with the same type of cell. The new approach described here involves the use of single cells of a mammalian cell line (BC3H1), containing muscle-type acetylcholine receptors, and the rapid introduction of neurotransmitter to the cell surface. The rapid delivery was achieved by converting a previously synthesized photolabile precursor of carbamoylcholine to carbamoylcholine, a stable amino-group-containing analogue of acetylcholine, with a single laser pulse and an observed photolysis rate of 7300 s-1. The resultant opening of the receptor channels creates a transmembrane current, which was measured in order to determine the rate constant for the formation of the open channel, k(op), the rate constant for channel closing, k(cl), and the dissociation constant of the receptor site controlling channel opening, K(l), the values of which were found to be 9400 s-1, 580 s-1, and 210-mu-M, respectively. Two of these constants, k(cl) and K(l), were also measured by two independent methods, and good agreement was observed. The value of k(op) is of interest because (i) it determines the rate at which signals can be transmitted between cells and (ii) signal transmission is determined by the concentration of receptors in the open-channel form. At any given concentration of neurotransmitter, the fraction of receptors in the open-channel form may be calculated from the values of K(l), k(cl), and k(op). These constants can now all be determined for the acetylcholine receptor, and for other receptors that are specific for neurotransmitters containing an amino group, using the laser-pulse photolysis technique described.