Dynamics of the acetylcholine receptor pore at the gating transition state

Abstract

Neuromuscular acetylcholine receptors (AChRs) are ion channels that alternatively adopt stable conformations that either allow (open) or prohibit (closed) ionic conduction. We probed the dynamics of pore (M2) residues at the diliganded gating transition state by using single-channel kinetic and rate-equilibrium free energy relationship (⌽-value) analyses of mutant AChRs. The mutations were at the equatorial (9) position of the ␣, ␤, and subunits (n ‫؍‬ 15) or at sites between the equator and the extracellular domain in the ␣-subunit (n ‫؍‬ 8). We also studied AChRs having only one of the two ␣-subunits mutated. The results indicate that the ␣-subunit, like the ␦-subunit, has a region of flexure near the middle of M2, that the two ␣-subunits experience distinct energy barriers to gating at the equator (but not elsewhere), and that the collective subunit motions at the equator are asymmetric during the AChR gating isomerization.ion channels ͉ phi-value ͉ rate-equilibrium free energy relationship (REFER) analysis T he acetylcholine receptor (AChR) is an allosteric protein that couples a change in affinity for ligands at the two transmitter binding sites with a change in conductance of its pore (1-3). To understand the mechanism of AChR gating, it is important to illuminate the complex molecular motions that link the stable closed (C) and open (O) conformations. The pore-lining, M2 segment has long been recognized as a critical structure in AChR gating (4). In particular, the equatorial 9Ј position (see Fig. 1 A) is a highly conserved Leu (in all five subunits) that forms a flexible region (a ''kink'') in the otherwise helical segment (5-7). It has been suggested that the equatorial region of M2 serves as the main barrier to ion permeation in the closed-channel conformation (5, 8), whereas substituted-Cys-accessibility studies place the ''gate'' closer to the cytoplasmic limit of M2 (9). Mutations at the M2 9Ј position have been shown to left-shift the whole-cell dose-response curve and cause a substantial increase in open channel lifetimes (10-13) and also affect desensitization (11,14). However, the effects of these 9Ј mutations on gating have not been analyzed at the level of equilibrium and rate constants.There is a spatial gradient in the extent to which AChR mutations that change the gating equilibrium constant alter the opening vs. the closing rate constant (15, 16). The closing rate constant is increasingly affected as one proceeds down the length of the protein, from the transmitter binding sites to the cytoplasmic limit of the transmembrane domain. Further, residues appear to be organized into contiguous domains within which all members have a similar behavior with regard to the effect of mutations on the opening vs. the closing rate constant (17). In some cases, these domains overlap secondary structural elements (e.g., helices), whereas in other cases such a direct correlation between function and structure is not apparent. The boundaries between these domains appear to be discrete (18).For a two-state r...

Principles underlying energetic coupling along an allosteric communication trajectory of a voltage-activated K + channel

“…Furthermore, our data are consistent with the ideas of Ranganathan and colleagues (4,5,19) explaining long-range communication in proteins through pathways of energetic connectivities. Further support for this mechanical view of signal transmission comes from studies on rate-energy relationships in the acetylcholine receptor channel (35,36). As such, the experimentally derived allosteric communication features presented here may advance the understanding of how long-range effects in proteins transpire.…”

Closed-state Cross-linking of Adjacent β1 Subunits in α1β1 GABAa Receptors via Introduced 6′ Cysteines

“…These may take the form of asymmetric movements (such as contrarotations of adjacent ␤ subunits) or as asynchronies in the movement of adjacent subunits during gating. Asymmetries in collective subunit motions have previously been observed for 9Ј residues during activation of the nAChR (21).…”

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

“…In a series of publications Auerbach and colleagues studied the relative timing of movements of domains of the nAChR initiated by ligand binding that result in the transitioning of channels from closed to open states. Upon acetylcholine binding the transmitter binding region involving loops A, B, and C moves first (24), loops 2 and 7 then move (25,26), and this is followed by the almost simultaneous movements of the TM2-3 linker region (26) and TM2 (4,27). It is believed that a "Brownian conformational wave" involving sequential movements of portions of receptor subunits ultimately links ligand binding to channel opening.…”