Channel proteins circumvent the enormous energetic barrier to ion transport imposed by the cell membrane and are essential to all life forms. When a channel activates, permeant ions flow passively down their electrochemical gradients, changing the membrane potential and allowing communication between extra- and intra-cellular environments. The selectivity of ion transport depends on the type of channel, which can be non-selective, charge-selective or ion-selective, suggesting a diversity of mechanisms underlying transport. Present day atomic structures of ion channels allow unprecedented studies of ion transport using computational approaches. Here we use all-atom molecular dynamics (MD) simulations to study transport of single cations through the charge-selective channel of the nicotinic acetylcholine receptor (nAChR) from the motor endplate.
The nAChR is a hetero-pentamer of 250 kD, and contains an intrinsic channel pore that triggers flow of cations in response to nerve-released ACh. The channel selects for cations, mainly according to size, and is formed by α-helices from the second transmembrane domains (TMD2) of each of the five subunits. Selectivity for cations is achieved by anionic residues located on either side of the channel mouth [1
] and in fenestrated structures in the cytoplasmic domain [2
], which stabilize cations and concentrate them relative to bulk solution. Hydrophobic residues extending from TMD2 line the narrow region of the nAChR channel [3
], suggesting that the hydration shell around the ion is maintained as the ion passes through. The availability of a 4 Å resolution structural model of the Torpedo
] has brought the atomic-scale mechanism of ion transport under intensive investigation [5
MD simulations of a simplified channel, composed of only TMD2 domains embedded in a bilayer-mimetic slab, revealed that although water filled the channel along its entire length, ions did not enter [7
]. In simulations containing the entire pore domain in an explicit lipid bilayer, water and ions were excluded from the narrow region of the channel [8
]. However, manually widening the pore radius by 1.5 Å allowed penetration of both water and ions, giving an ion transport rate approaching that expected from the single channel current amplitude. Although neither of these studies included the effect of membrane potential, they concluded that the cryo-electron microscopic structure of the Torpedo
nAChR is in the non-conducting, inactive state.
Analogous MD simulations have been conducted on the mechano-sensitive channel of small conductance (MscS), which like the nAChR, also contains a hydrophobic pore. The initial simulations suggested that the x-ray structure of MscS was in the non-conducting state because water was excluded from the narrow hydrophobic region during the majority of the simulation [9
]. However, application of a membrane potential promoted water entry, increased the channel radius, and produced ion transport at rates approaching those measured experimentally [10
]. These later simulations suggest that membrane potential facilitates entry of water, promoting ion transport, and that the x-ray structure of MscS is closer to a conducting than a non-conducting state.
Here, we generate a homology model of the nAChR found at the human adult motor endplate, embed it in explicit lipids and solvate it with water and ions. We apply a trans-membrane electric field to mimic the cell membrane potential, and perform all-atom MD simulations with and without restraints of the protein structure. Our results reveal transport of single cations, and show that the transport depends on hydration of the channel, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure.