End‐plate channel opening and the kinetics of quinacrine (mepacrine) block.

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Abstract
1. The effects of quinacrine on the relaxation of the agonist‐induced currents in response to a voltage step were investigated at voltage clamped frog end‐plates. A fast perfusion technique allowed the application of known concentrations of the agonist acetylcholine (ACh) or carbachol to end‐plate viewed with Nomarski optics. 2. In the presence of quinacrine, and in response to a hyperpolarizing voltage jump, an agonist‐induced current shows a fast initial relaxational increase and then relaxes slowly back to a new equilibrium level. 3. The slow relaxation can be described by a single exponential with a time constant tau s . tau s gets smaller at increasing quinacrine concentrations (0‐‐2 microM) and the decay rate constant, 1/tau s, increases linearly with quinacrine concentration. Increasing agonist concentration reduces tau s, in a manner dependent on the nature of the agonist. Tau s is markedly lengthened at more hyperpolarized potential, but this voltage effect gets less at higher concentrations of agonist. 4. These data suggest a slow voltage dependent blockage of open end‐plate channels by quinacrine. The binding rate constant of quinacrine is estimated as 10(8) M‐1 s‐1, and the voltage dependent, backward rate constant, as 5 s‐1 at ‐60 mV and 1 s‐1 at ‐140 mV. These values are in fair agreement with those obtained from the analysis presented in the preceding paper. 5. The agonist concentration dependence of the blocking kinetics is compatible with a simple model for channel opening. In this model, independent sequential binding of two agonist molecules leads to an isomerization of the receptor. The intrinsic binding constant of ACh is estimated to be around 20 microM, and for carbachol around 200 microM. Distinct isomerization constants could lead to a maximal activation of 70% of the available channels by ACh, and only 40% by carbachol. 6. An example of a possible interaction in between quinacrine block and desensitization is shown. At the break of an hyperpolarizing jump which has increased quinacrine blockade, a transient increase in the synaptic current is observed with apparently a temporary reduction of the desensitization.