Activation of acetylcholine receptor channels by covalently bound agonists in cultured rat myoballs.

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Abstract
1. Kinetic and equilibrium aspects of receptor activation by two irreversibly bound (''tethered'') agonists, QBr and bromoacetylcholine (BrACh), were examined in cultured embryonic rat muscle. Myoballs were treated with dithiothreitol (2 mM), washed, exposed to BrACh or QBr, and then washed again. Voltage-clamp recordings were made both in the whole-cell mode and with excised outside-out patches at 15.degree.C. 2. Whole-cell voltage-jump relaxations resembled those observed with reversibly bound agonists. The relaxation time constants were 5 ms for tethered QBr and 10 ms for tethered BrACh (-100 mV, 15.degree.C). At more positive membrane potentials, the relaxation rate constants increased and the conductance decreased. 3. Whole-cell light-flash relaxations with tethered QBr were also studied. The conductance was increased and decreased respectively, by cis .fwdarw. trans and trans .fwdarw. cis photoisomerizations. The relaxation time constants equalled those for voltage jumps. 4. The functional stoichiometry of tethered QBr was investigated by studying the relaxations in response to light flashes that produced known changes in the mole fractions of the two isomers. It is concluded that the open state of each receptor channel is controlled by the isomeric state of a single tethered QBr molecule. 5. In single-channel recordings, tethered agonists opened channels with the same conductance as reversibly bound agonists (30 pS at 15 .degree.C and -100 mV). More than 80% of the conductance was contributed by a population of openings with an average burst duration (lifetime) of 5 ms for QBr and 10 ms for BrACh. Thus the single-channel and macroscopic currents seem to be dominated by the same type of channel; these are presumably monoliganded receptors. 6. About 30% of the openings belonged to a population with an average lifetime of about 0.5 ms. This population contributed less than 5% of the conductance. There were also more long openings (< 50 ms) than expected from a simple exponential distribution. A few patches from BrACH-treated cells showed openings with a conductance of 45 pS (-100 mV) and an average duration of .apprx. 2 ms. 7. These data allow one to assess whether the agonist-receptor binding step plays a role in generating the brief openings. The main population of openings (burst durations 5 ms with QBr and 10 ms with BrACh) seem to be contributed by monoliganded receptors. One can therefore rule out the hypothesis that the brief channels arise exclusively from mono- and biliganded receptors, respectively.