To test our present quantitative knowledge of nicotinic transmission, we reconstruct the postsynaptic conductance change that results after a presynaptic nerve terminal liberates a quantum of acetylcholine (ACh) into the synaptic cleft. The theory assumes that ACh appears suddenly in the cleft and that is subsequent fate is determined by radial diffusion, by enzymatic hydrolysis, and by binding to receptors. Each receptor has one channel and two ACh binding sites; the channel opens when both sites are occupied and the rate-limiting step id the binding and dissociation of the second ACh molecule. The calculations reproduce the experimentally measured growth phase (200 microseconds), peak number of open channels (2,000), and exponential decay phase. The time constant of the decay phase exceeds the channel duration by approximately equal to 20%. The normal event is highly localized: at the peak, two-thirds of the open channels are within an area of 0.15 micrometer 2. This represents 75% of the available channels within this area. The model also simulates voltage and temperature dependence and effects of inactivating esterase and receptors. The calculations show that in the absence of esterase, transmitter is buffered by binding to receptors and the postsynaptic response can be potentiated.

After disulphide bonds are reduced with dithiothreitol, trans-3-(a-bromomethyl)-3'-[a-(t rimethylammonium) methyl]azobenzene(trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this "tethered agonist" shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 btM carbachol. Voltage-jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 #M carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-QBr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to "open-channel blockade" by QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3',bis-[a-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis ~ trans photoisomerizations and decreased by trans --* dsphotoisomerizations. As with Bis-Q, light-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered cis-QBr have a channel duration severalfold briefer than with the tethered trans isomer. By comparing the agonist-induced conductance with the cis/trans ratio, we conclude that each channel's activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. J. GEN. PHYSIOL. ~) The Rockefeller University Press

In these experiments, agonist-induced conductance is measured while a sudden perturbation is produced at the agonist-receptor binding site. A voltage-clamped Electrophorus electroplaque is exposed to trans-Bis-Q, a potent agonist. Some channels are open; these receptors have bound agonist molecules. A light flash isomerizes 3(-35)% of the trans-Bis-Q molecules to their cis form, a far poorer agonist. This causes a rapid decrease of membrane conductance (phase 1), followed by a slower increase (phase 2). Phase 1 has the amplitude and wavelength dependence expected if the channel closes within 100 mus after a single bound trans-Bis-Q is isomerized, and if the photochemistry of bound Bis-Q resembles that in solution. Therefore, the receptor channel responds rapidly, and with a hundred-fold greater closing rate, after this change in the structure of a bound ligand. Phase 2 (the conductance increase) seems to represent the relaxation back toward equilibrium after phase 1, because (a) phase 2 has the same time constant (1(-5) ms) as a voltage- or concentration-jump relaxation under identical conditions; and (b) phase 2 is smaller if the flash has led to a net decrease in (trans-Bis-Q). Still slower signals follow: phase 3, a decrease of conductance (time constant 5(-10 ms); and phase 4, an equal and opposite increase (several seconds). Phase 3 is abolished by curare and does not depend on the history of the membrane voltage. We consider several mechanisms for phases 3 and 4.

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