Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Significance Our mutagenesis work on the ring of glutamates in the charge-selectivity filter region of the muscle nicotinic acetylcholine receptor led us to propose that the rate at which ions permeate through the open channel depends not only on the number of these glutamates, but also on the conformation of their side chains. Because our inferences were made on the basis of electrophysiological observations, however, we decided to test the plausibility of these ideas using computer simulations. Remarkably, the simulations gave ample credence to all aspects of our proposal and allowed us to gain insight into the effect of specific glutamate rotamers on single-channel conductance. On the basis of single-channel currents recorded from the muscle nicotinic acetylcholine receptor (AChR), we have recently hypothesized that the conformation adopted by the glutamate side chains at the first turn of the pore-lining α-helices is a key determinant of the rate of ion permeation. In this paper, we set out to test these ideas within a framework of atomic detail and stereochemical rigor by conducting all-atom molecular dynamics and Brownian dynamics simulations on an extensively validated model of the open-channel muscle AChR. Our simulations provided ample support to the notion that the different rotamers of these glutamates partition into two classes that differ markedly in their ability to catalyze ion conduction, and that the conformations of the four wild-type glutamates are such that two of them “fall” in each rotamer class. Moreover, the simulations allowed us to identify the mm (χ1 ≅ –60°; χ2 ≅ –60°) and tp (χ1 ≅ 180°; χ2 ≅ +60°) rotamers as the likely conduction-catalyzing conformations of the AChR’s selectivity-filter glutamates. More generally, our work shows an example of how experimental benchmarks can guide molecular simulations into providing a type of structural and mechanistic insight that seems otherwise unattainable.