Activation and Block of the Adult Muscle-Type Nicotinic Receptor by Physostigmine: Single-Channel Studies

Abstract

The plant-derived acetylcholinesterase inhibitor physostigmine has previously been shown to act on the nicotinic acetylcholine receptor (nAChR) causing either direct activation or potentiation of currents elicited by low concentrations of nicotinic agonists, or, at higher concentrations, channel block. We examined mouse adult-type muscle nAChR activation by physostigmine and found that channel activation by physostigmine exhibits many characteristics common with channel activity elicited by nicotinic agonists. Single-channel conductance was indistinguishable, and mutants known to slow channel closing in the presence of nicotinic agonists had a similar effect in the presence of physostigmine. However, physostigmine is a very inefficacious agonist. The presence of physostigmine did not alter the effective opening rate for a subsaturating dosage of carbachol, suggesting that physostigmine does not interact with the nicotinic agonist binding site. Mutations to a residue (␣Lys125) previously identified as part of the putative binding site for physostigmine reduced the duration of openings elicited by physostigmine, but the effects were generally small and, in most cases, nonsignificant. At higher concentrations, physostigmine blocked channel activity. Block manifested as a reduction in the mean open time and the emergence of a closed state, with a mean duration of 3 to 7 ms. The properties of block were consistent with two equivalent blocking sites per receptor with microscopic binding and unbinding rate constants for physostigmine of 20 M Ϫ1 s Ϫ1 and 450 s Ϫ1 (K D ϭ 23 M). These observations indicate that physostigmine is able to activate muscle nAChR by interacting with a site other than the nicotinic ligand binding site.

Positive and negative modulation of nicotinic receptors

“…Nevertheless, the orientation of physostigmine in the a4b2 AChR is slightly different. Although these results suggest a role of a-Lys in the allosteric binding of physostigmine and galantamine, the mutation a7-Lys123Gly perturbs neither nicotine activation nor galantamine potentiation (Luttmann et al, 2009), whereas mutations on a1-Lys125 reduce the direct stimulatory effect elicited by physostigmine, but not by carbachol, on muscle AChRs (Militante et al, 2008). It has been postulated that this site, although not located within the ion channel, is involved in the noncompetitive inhibition mediated by physostigmine (and galantamine) at high concentrations (> 10 mM) (Luttmann et al, 2009).…”

“…It has been postulated that this site, although not located within the ion channel, is involved in the noncompetitive inhibition mediated by physostigmine (and galantamine) at high concentrations (> 10 mM) (Luttmann et al, 2009). However, this contradicts the evidence, suggesting that this site is involved in the agonistic action of physostigmine, and it is more likely that the noncompetitive inhibition is elicited by ion channel blocking (Militante et al, 2008).…”

Cytoplasmic Ca2+ concentration changes evoked by muscarinic cholinergic stimulation in primary and metastatic melanoma cell lines

“…Nevertheless, the application of CCh (1 mmol/l) raises a question about the possible involvement of the nicotinic acetylcholine receptors as well, as in this concentration CCh was reported to act on these receptors, too [29]. Moreover, the expression of certain nicotinic acetylcholine receptor subtypes was detected in various cells of the skin (for reviews see [27,28]), and to make the situation even more complex, atropine was reported to block various nicotinic receptor subtypes [30].…”

Cytoplasmic Ca2+ concentration changes evoked by cholinergic stimulation in primary astrocyte cultures prepared from the rat cochlear nucleus

“…Although the above findings indicated the decisive role of the muscarinic cholinergic receptors, it was important to check the possible involvement of the nicotinic ACh receptors, as 1 mM CCh was reported to activate these receptors, too (Militante et al, 2008). Fig.…”

“…In the rat CN both the presence of nicotinic ACh receptors (Happe and Morley, 1998) and their CCh sensitivity (in single channel measurements; Militante et al, 2008) have been documented. It seems, therefore, that nicotinic receptors may have functional significance in the rat CN, but our specific experiments pointed out that muscarinic receptors may play a far more significant role in the genesis of the CCh-induced transients, at least under the experimental conditions applied in this work.…”