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

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801

“…The simplest mechanism we can think of that is consistent with these (33). The laser-pulse photolysis method was used as has been described in detail (23,24,27,28). Seventy micromolar carbamoylcholine was photolytically released from the 1-mM caged carbamoylcholine with which the cell was preequilibrated in the absence (Top) and presence (Bottom) of 500 M MK-801 or in the presence of both 500 M MK-801 and 2 M aptamer II-3 (Middle).…”

“…k op and kcl are the rate constants for channel opening and closing, respectively; ⌽ Ϫ1 (ϭkop͞kcl) is the channel-opening equilibrium constant (25, 43). The reactions shown occur in the microsecond-to-millisecond time region (13,(22)(23)(24)44). For clarity, the desensitization reaction, which in the case of the AChR occurs in the 100-to 500-ms time region (44,45), and the binding of the inhibitor I to the unliganded receptor form are not shown.…”

“…Here we demonstrate that by using transient kinetic techniques (22)(23)(24)(25)(26), one can obtain additional information about the inhibition mechanism.…”

“…Recently, presteady-state kinetic techniques that are suitable for measuring receptor-mediated reactions on cell surfaces in the millisecond-to-microsecond time region were developed (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). The time resolution of the laser-pulse photolysis technique (23)(24)(25)(26)) is sufficient to investigate the reaction before the channel has opened. One can, therefore, obtain information about the rate constants of channel opening (k op ) and closing (k cl ), the equilibrium constants for channel opening (⌽ Ϫ1 ) and neurotransmitter binding to the site controlling channel opening (K 1 ) (reviewed in ref.…”

“…A standard concentration (100 M) of carbamoylcholine was applied by using the cell-flow technique (22) before and after each photolysis experiment. The current amplitudes corrected for receptor desensitization (22,35) were compared with the current amplitudes obtained in the laserpulse photolysis experiments and with standard curves that allow one to relate current amplitudes to carbamoylcholine concentration (24). For each concentration of carbamoylcholine used, control experiments without inhibitor were performed, and the current amplitude corrected for receptor desensitization was taken as the 100% response.…”