Identification of Acetylcholine Receptor Channel-Lining Residues in the M1 Segment of the .alpha.-Subunit

Akabas, Myles H.; Karlin, Arthur

doi:10.1021/bi00039a002

Cited by 183 publications

(149 citation statements)

References 33 publications

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“…Of 106 Cys-substitution mutants expressed in Xenopus oocytes or HEK 293 cells, 105 appeared at the cell surface and conducted cations when activated by ACh, 38 reacted with at least one charged methanethiosulfonate, and 24 reacted with appreciably different rate constants in the resting and open states of the receptor (52,(58)(59)(60)(61). The reactivities of the substituted Cys were different in the resting and open states in the N-terminal third of the M1 segments, close to the extracellular side of the membrane, and along the entire length of the M2 segments.…”

mentioning

confidence: 99%

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

Wilson¹

2001

Proceedings of the National Academy of Sciences

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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...

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mentioning

confidence: 99%

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

Wilson¹

2001

Proceedings of the National Academy of Sciences

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“…This supports previous photolabeling studies (34,35) as well as mutagenesis studies that demonstrate mutations in this region increase receptor sensitivity and stabilize the open state of the pore (31, 36 -38). Clearly, previous substituted cysteine accessibility method studies and the data we have presented here favor a gate located closer to the cytoplasmic boundary of TM2 (8,25). We are a bit skeptical about assigning a gate location, even an approximate one, from a substituted cysteine accessibility method analysis, because many mutations in the pore can destabilize the closed state relative to the open state and produce agonist-independent openings.…”

Section: Discussionmentioning

confidence: 73%

“…Sitedirected mutagenesis studies, as well as cysteine scanning in all member of this receptor-operated family, have further supported this role for TM2 (6 -8). A cysteine scan of TM1 in the homologous nACh receptor demonstrated a stretch of accessible residues on the extracellular half, leading the authors to conclude that the extracellular third of TM1 is accessible by means of the pore and thus may contribute to the pore structure (25). A cysteine scan of TM3 of the GABA receptor ␣ subunit revealed two accessible residues near the extracellular end in the absence of GABA and three additional residues that were accessible in the presence of agonist.…”

Section: Discussionmentioning

confidence: 99%

“…Initial studies in the nACh and GABA receptors have demonstrated accessibility in the absence of agonist near the cytoplasmic end of TM2 (7,25). More recent studies using reagents added to either side of the membrane identified a barrier for accessibility in the absence of agonist in the ␣ subunit of the nACh receptor between Gly 240 and Thr 244 (32).…”

Section: Discussionmentioning

confidence: 99%

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Evidence that the TM1-TM2 Loop Contributes to the ρ1 GABA Receptor Pore

Filippova

Wotring

Weiss

2004

Journal of Biological Chemistry

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Considerable evidence indicates the second transmembrane domain (TM2) of the ␥-aminobutyric acid (GABA) receptor lines the integral ion pore. To further delineate the structures that constitute the ion pore and selectivity filter of the 1 GABA receptor, we used the substituted cysteine accessibility method with charged reagents to identify anion-and cation-accessible surfaces. Twenty-one consecutive residues were mutated to cysteine, one at a time, in the presumed intracellular end of the first transmembrane domain (TM1; Ala 271 -Met 276 ), the entire linker connecting TM1 to TM2 (Leu 277 -Arg 287 ), and the presumed intracellular end of TM2 (Ala 288 -Ala 291 ). Positively (MTSEA ؉ ) and negatively (pCMBS ؊ ) charged sulfhydryl reagents, as well as Cd 2؉ , were added extracellularly to test accessibility of the engineered cysteines. Four of the mutants, all at the intracellular end of TM2 (R287C, V289C, P290C, A291C), were accessible to positively charged reagents, whereas seven mutants (A271C, T272C, L277C, W279C, V280C, P290C, A291C) were functionally modified by negatively charged pCMBS ؊ . These seven modified residues were at the intracellular end of TM2, in the TM1-TM2 linker, and at the intracellular end of TM1. In nearly all cases (excluding P290C), the rate and the degree of modification were state-dependent, with greater accessibility in the presence of agonist. Select cysteine mutants were combined with a point mutation (A291E) that converted the pore from chloride-to non-selective. In this case, positively charged reagents could modify residues in the TM1-TM2 linker (Leu 277 and Val 280 ), supporting the notion that the modifying reagents were reaching their target through the pore. Taken together, our results suggest that, up to its intracellular end, the TM2 domain is not charge selective. In addition, we propose that the TM1-TM2 linker and the intracellular end of TM1 are along the pathway of the permeating ion. These findings may lend new insights into the structure of the GABA receptor pore.Ligand-gated ion channels play a fundamental role in neuronal communication. The ␥-aminobutyric acid (GABA) 1 receptor is the major inhibitory neurotransmitter-activated ion channel in mammalian brain and is an important target for a variety of clinically prescribed therapeutic compounds. Detailed knowledge of its structure and function can provide important information for understanding the fundamentals of neuronal communication, the treatment of neurological disorders, and the design of therapeutic compounds that target the GABA receptor.The family of ligand-activated ion channels that includes the GABA, nicotinic acetylcholine (nACh), glycine, and serotonin type 3 receptors (1-5) is composed of a group of proteins with diverse functional properties but homologous structure. This structure includes four predicted transmembrane domains (TM1-TM4), a large extracellular amino-terminal domain containing the agonist-binding site, and a large intracellular linker between TM3 and TM4 that has been suggested to pla...

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“…Cysteine-scanning mutagenesis, combined with cysteine-reactive compounds, has been a powerful and extensively used technique to examine nAChRs [253][254][255]. Another powerful experimental approach is the incorporation of unnatural amino acids into…”

Section: Expression Of Nachrs Altered By Site-directed Mutagenesismentioning

confidence: 99%

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

Millar

2009

Biochemical Pharmacology

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Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of neurotransmitter-gated ion channels, a family that also includes receptors for γ-aminobutyric acid, glycine and 5-hydroxytryptamine. In humans, nAChRs have been implicated in several neurological and psychiatric disorders and are major targets for pharmaceutical drug discovery. In addition, nAChRs are important targets for neuroactive pesticides in insects and in other invertebrates. Historically, nAChRs have been one of the most intensively studied families of neurotransmitter receptors. They were the first neurotransmitter receptors to be biochemically purified and the first to be characterized by molecular cloning and heterologous expression. Although much has been learnt from studies of native nAChRs, the expression of recombinant nAChRs has provided dramatic advances in the characterization of these important receptors. This review will provide a brief history of the characterization of nAChRs by heterologous expression. It will focus, in particular, upon studies of recombinant nAChRs, work that has been conducted by many hundreds of scientists during a period of almost 30 years since the molecular cloning of nAChR subunits in the early 1980's.

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Identification of Acetylcholine Receptor Channel-Lining Residues in the M1 Segment of the .alpha.-Subunit

Cited by 183 publications

References 33 publications

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

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

Evidence that the TM1-TM2 Loop Contributes to the ρ1 GABA Receptor Pore

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

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