Dose‐response of acetylcholine receptor channels opened by a flash‐activated agonist in voltage‐clamped rat myoballs.

Abstract

SUMMARY1. Whole-cell or single-channel currents through acetylcholine (ACh) receptor channels were studied in voltage-clamped rat myoballs or in excised membrane patches from myoballs. The recording pipette contained CsC1 to suppress outward currents, and tetrodotoxin was used to help suppress Na+ currents.2. To minimize problems associated with bath applied agonists, myoballs were bathed in a solution containing the inactive (cis) isomer of the photo-isomerizable azobenzene derivative, Bis-Q. Calibrated light flashes of varying intensity were presented to produce concentration jumps of agonist, trans-Bis-Q. The resulting whole-cell current relaxations through ACh channels approach a steady state along an exponential time course, then decline as the newly created agonist diffuses away over the next few seconds.3. The dose-response relationship was inferred from Hill (double-log) plots for myoballs bathed in 500 nM-cis-Bis-Q at three membrane potentials. At low agonist concentrations (< 300nM-trans-Bis-Q), the slope of the Hill plot averaged 1'62 at -150 mV, 1-89 at -100 mV, and 2-05 at + 80 mV. These results are consistent with an apparent agonist affinity constant that decreases with membrane depolarization and shifts the responses further down on the dose-response curve.4. When the myoballs were bathed in higher concentrations ofcis-Bis-Q (1 -5-20 /LM), the slope of the Hill plot was reduced at all membrane potentials, although it was still closer to two at positive potentials. This is expected from the known sigmoid shape of the dose-response relation. The shallow dependence of the Hill slope on agonist concentration suggests the presence of negative cooperativity in the over-all binding of agonist molecules.5. Following treatment of the membrane with dithiothreitol to reduce disulphide groups, the Hill slope for the reversibly bound agonist, trans-Bis-Q, remained near two.6. The kinetics of currents at hyperpolarized membrane potentials became complicated at higher agonist concentrations in a manner that was consistent with L. D. CHABALA, A. M. GURNEY AND H. A. LESTERopen-channel block by tranw-Bis-Q; the currents showed a slow secondary increase in conductance.7. Averaged single-channel recordings at higher agonist concentrations resemble macroscopic relaxations under comparable conditions. Furthermore, those recordings also suggested that open channels are blocked by trans-Bis-Q at concentrations > 2,um; the block depends strongly on membrane potential and increases with hyperpolarization. Currents at positive membrane potentials showed no evidence of open-channel block.8. In the presence of 20 tM-Ci8-Bis-Q, the probability of a channel being open was constant from one episode to the next for up to several hundred flash-induced agonist concentration jumps (0 to 10 6/tM-trans-Bis-Q).9. Fractional activation of single ion channels at the highest agonist concentration tested (20 1M-cis-Bis-Q) was small, which agrees with the observation that whole-cell currents, under similar conditions, are still in the low...

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

“…1 A, for example, membrane properties of a myoball were monitored during pressure application of QBr. The increased conductance (roughly twice the resting conductance at -100 mV) was stable for at least 20 min after withdrawal of the pressure pipette, whereas with similar pressure application of reversibly bound agonists, the agonist-induced conductance persisted for only a few seconds after withdrawal, until the agonist diffused away (see also Chabala et al 1986). …”

“…2 B). These signals partially resemble light-flash relaxations with the reversibly bound agonist, Bis-Q, but there is an important difference (Chabala, Gurney & Lester, 1985;Chabala et al 1986). The Bis-Q is present in the bathing solution, so that within a few seconds after the flash, the newly isomerized molecules undergo diffusive exchange for fresh non-irradiated molecules.…”

“…Most procedures, including the preparation of primary cultures of rat myoballs and the preparation of solutions, were identical to those previously described (Chabala, Gurney & Lester, 1986). …”

“…The left-hand panel shows data for a myoball bathed in 500 nM-Bis-Q; the right-hand panel shows the data on QBr from the cell of A. Although the experiment with Bis-Q gives a slope of about 2 on double-logarithmic coordinates (see also Chabala et al 1986), the value for tethered QBr is 0 97; and for a total of four complete experiments, the average slope was 1I01+ 0 1. The doublelogarithmic plots thus give further support to the hypothesis of receptor activation by a single tethered trans-QBr molecule.…”

“…The extent of photoisomerization of tethered QBr can be varied by inserting neutral-density filters into the beam (Chabala et al 1986). Fig.…”