The outer leucine ring of the ion pore of the nicotinic acetylcholine receptor is an important determinant for channel gating
Authors:   , Chen Zhaoming , White Michael M 
Published: July 2024

The muscle-type nicotinic acetylcholine receptor (nAChR) is located the muscle end plate and is responsible for muscle contraction. It is a pentameric complex having a stoichiometry of [alpha]₂[beta][gamma][delta] for fetal muscle and [alpha]₂[beta][epsilon][delta] for adult muscle. Each subunit contains four transmembrane domains named M1, M2, M3 and M4. The M2 segments from the five subunits come together to constitute an ion pore, in which five amino acids of same level of M2 segment interact with each other to form a series of functional 'rings' responsible for channel gating. The 'outer leucine ring' formed by leucine/valine residues at position 17[prime] is the focus of this project. Replacement of amino acid with different side chain on position 17[prime] of [epsilon] subunit reduced the EC₅₀ of nAChR for ACh, indicating that the side chain on position 17[prime] of the [epsilon] subunit is important for the normal function of the ion channel. This effect was also observed by glutamic acid substitution on aligned amino acids of outer leucine ring in other [alpha], [beta], and [delta] subunits. The EC₅₀ reduction of the [delta] subunit substitution was twice as much as any other single substitution. The EC₅₀ decreased further as the number of subunit substitutions increased, and [delta] subunit always had the lowest EC₅₀ among single, double and triple substitutions. The change in free energy of multiple substitutions was similar to the linear combination of the change in free energy for individual ones. These results indicated that the five residues of outer leucine ring work independently, additively, though not all evenly. The similar Hill coefficients between wild type and mutants implied that same number of agonist molecules was required for receptor activation. We can not record the whole cell currents if more than three substituted subunits were included in nAChR. It is due to little or no intact receptor protein being expressed on the cell surface. Single channel recording demonstrated that the increased potency of ACh for mutant nAChR is attributed to prolonged mean open time not to a change in conductance. It suggested the existence of more stable open state of mutated ion channel (enhanced agonist efficacy) and more contribution of [delta] subunit to the channel gating, as the alteration of gating at macroscopic level was mirrored at the microscopic level. As little or no change in agonist binding affinity was observed for the mutant nAChR. In addition, equivalent shift of ratio in the microscopic EC₅₀ over macroscopic EC₅₀ among wild type and mutants implied that the alteration of closed rate constant [alpha] was sufficient to explain the changes of EC₅₀ measured by whole cell recording. It is proposed that the five residues of outer leucine ring, together with other stratified rings inside ion pore, approach one another upon the leave of ACh and move apart in the presence of agonist. These rings may therefore gate the ion pore to control the ion movement. The impairment of these rings via the introduction of mutation may cause ion channel leaky, widen and slow closure.