Binding-gating coupling in a nondesensitizing α7 nicotinic receptor A single channel pharmacological study

The highly conserved alpha Lys145 has been suggested to play an important role in the early steps of activation of the nicotinic acetylcholine receptor (nAChR) by acetylcholine. Both macroscopic and single channel currents were recorded in the slowly desensitizing mutants L248T- and K145A-L248T-alpha 7 receptors expressed in Xenopus oocytes. On ACh-evoked currents, substitution of Lys145 by alanine showed the same effects that in wild type receptors: moderately decreased gating function and a more-than-expected loss of ACh potency, thus validating the experimental model. Single channel analysis quantitatively agreed with macroscopic data and revealed that impaired gating function in the double mutant alpha 7K145A/L248T is the consequence of a slower opening rate, beta. Several nicotinic agonists were also studied, showing important features. Particularly, dimethylphenylpiperazinium, (DMPP), acting as an antagonist in alpha 7K145A, became a full agonist in alpha 7K145A/L248T. Single channel analysis of DMPP-evoked currents showed effects of Lys145 removal similar to those observed with ACh. Data suggest that alpha 7Lys145 facilitates the early steps of channel activation. Moreover, the slowly desensitizing mutant alpha 7L248T could be an interesting tool for the study of channel activation in alpha 7 receptors. Nevertheless, its extensively altered pharmacology precludes the simple extrapolation of pharmacological data obtained in singly mutated alpha 7 receptors. (c) 2008 Elsevier B.V. All rights reserved.