Analysis of cyclic and acyclic nicotinic cholinergic agonists using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy

Abstract

The relationship between the structure and function of a series of nicotinic cholinergic agonists has been studied using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy. The cyclic compound 1,1-dimethyl-4-acetylpiperazinium iodide and its trifluoromethyl analogue (F3-PIP) interact with nicotinic acetylcholine receptors (nAChRs) from both Torpedo electroplaque and BC3H-1 cells at lower concentrations than the acyclic derivatives, N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide and its fluorinated analogue (F3-TED). The magnitude of the difference in potencies depends on the type of measurement. In binding experiments, the differences between the two classes of compounds depends mainly on the conditions of the experiment. In measurements of the initial interaction with the nAChR, the PIP compounds have an affinity approximately one order of magnitude higher than that of the TED compounds. Longer incubations indicated that the PIP compounds were able to induce a time-dependent shift in receptor affinity consistent with desensitization, whereas the TED compounds were unable to induce such a shift. The activation of single channel currents by the cyclic compounds occurs at concentrations approximately two orders of magnitude lower than for the acyclic compounds, but the TED compounds exhibit a larger degree of channel blockade than the PIP compounds. Previous work (McGroddy, K.A., and R.E. Oswald. 1992. Biophys. J. 64:314-324) has shown that the TED compounds can exist in two energetically distinct conformational states related by an isomerization of the amide bond. 19F nuclear magnetic resonance experiments suggest that the higher energy population of the TED compounds may interact preferentially with the ACh binding sites on the nAChRs and that a significant fraction of the difference between the initial affinity of the PIP and TED compounds may be accounted for by the predominance in solution of a conformational state less able to interact with the ACh binding sites on nAChRs.

Block of Quantal End-Plate Currents of Mouse Muscle by Physostigmine and Procaine

“…Such flickering block also has been seen with high ACh concentrations in hyperpolarized patches, with blocking rates in the range of 5 ⅐ 10 7 M Ϫ1 s Ϫ1 and unblocking rates of 5 ⅐ 10 4 s Ϫ1 . These unblocking rates correspond to ''flickering,'' short closings of the channel with an average duration of 20 s (McGroddy et al 1993;Ogden and Colquhoun 1985;Parzefall et al 1998;Sine and Steinbach 1984;Sine et al 1990).…”