Inaugural Article: Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

Abstract

The nicotinic acetylcholine (ACh) receptors cycle among classes of nonconducting resting states, conducting open states, and nonconducting desensitized states. We previously probed the structure of the mouse-muscle ACh receptor channel in the resting state obtained in the absence of agonist and in the open states obtained after brief exposure to ACh. We now have probed the structure in the stable desensitized state obtained after many minutes of exposure to ACh. Muscle-type receptor has the subunit composition ␣2␤␥␦. Each subunit has four membrane-spanning segments, M1-M4. The channel lumen in the membrane domain is lined largely by M2 and to a lesser extent by M1 from each of the subunits. We determined the rates of reaction of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl methanethiosulfonate with cysteines substituted for residues in ␣M2 and the ␣M1-M2 loop in the desensitized state and compared these rates to rates previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states of the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-aminoethyl methanethiosulfonate, we previously located the closed gate in the resting state between ␣G240 and ␣T244, in the predicted M1-M2 loop at the intracellular end of M2. Now, we have located the closed gate in the stable desensitized state between ␣G240 and ␣L251. The gate in the desensitized state includes the resting state gate and an extension further into M2.T he nicotinic acetylcholine (ACh) receptors cycle among three classes of functional states: resting, open, and desensitized (1). The receptor is conducting in the open state and nonconducting in the resting and desensitized states. The resting state is the most stable state when no agonist is bound, whereas the desensitized state is the most stable state when agonist is bound. Muscle-type ACh receptors have two ACh binding sites corresponding to the two ␣ subunits in the pentameric complex (Fig. 1, refs. 2 and 3). In the prevailing cycle of transitions, receptors in the resting state bind two molecules of ACh, isomerize to the open state, and, in the continued presence of ACh eventually desensitize. After the removal of free ACh and its dissociation from the binding sites, receptors in the desensitized state predominantly isomerize directly to the resting state. This cycle of activation, desensitization, and recovery has been extensively studied electrophysiologically (1, 4-7). The role of desensitization in cholinergic neurotransmission under normal physiological conditions is uncertain but is evident under some pathological conditions and in neurotransmission by other neurotransmitters (8).Desensitization occurs in stages (9). Receptor isomerizes to a transient, fast-onset desensitized state on the 0.1-to 10-s time scale and to a stable, slow-onset desensitized state on the 10-to 100-s time scale (10-17). The ACh affinities of the...

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

“…Nevertheless, both sample conditions provided consistent conformational changes. The changes observed in the transmembrane pore of ELIC from the resting state to a desensitized state agrees well with the GABA A Rβ3 structure (Miller and Aricescu, 2014) and is partially in line with several existing models (Auerbach and Akk, 1998; Keramidas and Lynch, 2013; Velisetty et al, 2012; Wilson and Karlin, 2001). The expanded extracellular portion of the pore was also observed in GLIC (Parikh et al, 2011; Velisetty and Chakrapani, 2012; Velisetty et al, 2012) and GABA A Rβ3 (Miller and Aricescu, 2014).…”

“…Although the desensitized state of pLGICs is functionally distinct from the resting and open states (Wilson and Karlin, 2001), the structural differences of a desensitized channel from those in the other two functional states are far less clear. The debate about what causes non-conductivity in the pore of desensitized pLGICs continues, despite the fact that several structural models have been proposed to describe pLGIC desensitization (Dellisanti et al, 2013; Keramidas and Lynch, 2013; Velisetty and Chakrapani, 2012; Wang and Lynch, 2011; Zhang et al, 2011).…”

Pore Structure of the Cys-loop Ligand-gated Ion Channels

“…For example, in the nAChR MTS reagents are able to access the closed pore and react with substituted cysteines as far as aThr 2 0 [26,27], which is certainly further than would be expected from the ELIC structure. It is also further than predicted from the Torpedo structure, but the desensitized state (after exposure to agonist for minutes) shows accessibility only as far as aLeu 9 0 [28]. Functional data also indicates that the extracellular half of the M2 region is a more dynamic structure than the lower, constricted part of the channel.…”

“…The results from SCAM studies have been used to infer the location of the gate by analysing the residues accessible to MTS reagents in the absence and then presence of agonist. As alluded to above, the gate in the nAChR appears to be between aGly-2 0 and aThr 2 0 in the closed state [26,27], but extending as far as aLeu 9 0 in the desensitized state [28]. The gate in both the GlyR and GABA A R is also likely to be further towards the intracellular pore than the ''hydrophobic girdle'' found in the Torpedo structure.…”

“…The first and most important of these comes from the substituted-cysteine accessibility method (SCAM), a technique used for scanning ion channel structures to identify water-accessible surfaces. This technique has been applied to the M2 region of nAChR [25][26][27][28], GABA A R [29,30], 5-HT 3A R [31,32] and GlyR [33] in previously published work. It is timely to reassess this data in the light of the new crystals structure.…”