Regulation of alpha7 nicotinic acetylcholine receptor function and pharmacology by amino acid sequence in the second transmembrane domain

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels with clearly defined physiological roles at the neuromuscular junction and in peripheral ganglia, and more mysterious roles in the mammalian brain, and non-neuronal tissues. The larger family of nicotinic receptors can be broadly categorized into three major subgroups based on subunit composition, anatomical distribution, and functional and pharmacological differences. These are the muscle-type receptors, the heteromeric neuronal receptors, and the homomeric receptors, typified by receptors composed of the alpha7 subunit.; Our studies demonstrate that a conferring of a functional phenotype can be accomplished by systematic substitution of amino acid sequence from the beta subunits of either muscle-type (beta1) or neuronal (beta2/beta4) nAChRs into homomeric receptors composed of mutant alpha7 nAChR subunits. Specifically, the alpha7 TM2 T6'F mutant shows properties similar to the muscle-type nAChR with regard to divalent ion permeability, current rectification, agonist concentration-dependent kinetics, sensitivity to succinycholine, and a lack of potentiation by 5-hydroxyindole. While a variety of muscle receptor-like properties are observed in the T6'F mutant, the alpha7 TM2 T6'S mutant, which has amino acid sequence identical to the neuronal beta2/beta4 subunit at this position, demonstrates significant kinetic similarities to neuronal nAChRs, but largely retains the pharmacology of the wild-type alpha7 receptor. At the single-channel level, the T6'S mutant has a unitary conductance similar to that reported for wild-type alpha7, but a vastly longer average open duration. Furthermore, channel burst activity indicates a significantly greater likelihood of channel opening in the sustained presence of agonist relative to wild-type.; The significant impact of these TM2 6' substitutions on a variety of functional aspects of the mutant alpha7 receptors suggests that amino acid sequence at this position contributes to several important features that distinguish the major nAChR subgroups from one another. Furthermore, the alpha7 TM2 T6'S mutant shows a kinetic gain of function in the absence of significant pharmacological differences from the wild-type alpha7 receptor. Thus it may provide the ability to observe agonist-evoked signals using contemporary high-throughput drug screening methods (where the wild-type receptor would be inhibited), implicating it as a potential tool for identifying alpha7-selective compounds.