Mechanism of hydrophobic gating in the acetylcholine receptor channel pore.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

Protein engineering and design in ion channels and receptors.

Acetylcholine receptors (AChRs) expressed at the neuromuscular junction synapses are typical allosteric proteins that shuttle between at least two stable conformational states: Closed (C) and Open (O). Agonist binding to their target sites on the receptor in the extracellular domain triggers a global C→O conformational change that results in an open channel pore that allows ion conduction. How the receptor senses the chemical signal of an agonist and communicates it to the channel pore, located ~50Šaway, are key to understanding the receptor function. AChRs are indispensable for muscle contraction and their neuronal homologues play critical roles in the nervous system function. In this chapter, using a combination of single channel patch-clamp, computational approaches, and genetic engineering, we elucidate the principles of design and engineering to quantify the fundamental thermodynamic parameters of AChRs that regulate ligand binding and channel opening. The receptor engineering principles outlined here for the neuromuscular AChRs are applicable to the broader class of ligand-gated ion channel proteins.