Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates

Stauffer, David A.; Karlin, Arthur

doi:10.1021/bi00188a013

Cited by 280 publications

(271 citation statements)

References 50 publications

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“…Consistent with this interpretation, MTSEA reduced ␥ more rapidly when applied directly onto the intracellular aspect of an inside-out patch. Using this approach, the rate of reduction of the amplitude of unitary events was still substantially slower than the rate of MTS modification of simple thiol compounds and easily accessible substituted Cys residues in the outer mouth of the Shaker B K ϩ channel (16,17). Such data suggest that MTSEA encounters a rate-limiting barrier en route to the MA 0Ј residue within the portal.…”

Section: Discussionmentioning

confidence: 94%

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Deeb¹,

Carland

Cooper

et al. 2007

Journal of Biological Chemistry

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Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates ␥, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.Cysteine loop (Cys loop) receptors encompass five families of gene products. Nicotinic-acetylcholine (nACh) 3 receptors, 5-hydroxytryptamine type-3 (5-HT 3 ) receptors, and Zn 2ϩ -activated channels conduct cations, whereas ␥-aminobutyric acid type A and glycine receptors conduct anions (1). Cys loop receptor subunits combine as pentamers forming a central ion pore that traverses the plasma membrane. Studies examining the role of residues comprising the pore-lining second transmembrane (TM2) helices of the nACh receptor led to the traditional view that this region of Cys loop receptors serves as the rate-limiting barrier to ion flux. Among the cationselective Cys loop receptor subunits, conserved acidic residues influence unitary conductance (␥) by forming concentric rings of negative charge positioned along the vertical axis of the central pore (2).Although the role of residues within the TM2 helix of Cys loop receptors in controlling ␥ is well established, recent studies demonstrate a similar function for residues located in the large cytoplasmic TM3-4 loop (1, 3, 4). Based on a 4.6 Å resolution cryoelectron microscopy image of the Torpedo marmorata nACh receptor, Miyazawa et al. (5) first speculated that amino acids in the TM3-4 loop form "transverse tunnels" that may contribute to the ion conduction pathway. Subsequent electrophysiological studies demonstrated that cytoplasmic residues within membrane-associated (MA) helices of the 5-HT 3 and ␣ 4 ␤ 2 nACh receptors are determinants of ␥ (3, 4). Arg 436 in the 5-HT 3A subunit at a position termed MA 0Ј is critical for maintaining the anomalously low ␥ (ϳ900 femtosiemens) that is a hallmark of the homomeric 5-HT 3A receptor. Furthermore, introduction of MA 0Ј Arg into ␣ 4 ␤ 2 nACh receptors halved ␥ (4).A 4 Å resolution model of the T. marmorata nACh receptor indicates that adjacent MA helices frame portals with apertures only slightly larger than partially hydrated permeant cations (6). Using this structure as a template, homology models of the 5-HT 3A and ␣ 4 ␤ 2 nACh receptors indicate that charged amino acids line their cytoplasmic portals (4).Although it is now clear that specific MA helix residues influence the ␥ of nACh and 5-HT 3 receptors, it remains to be determined whether their charge plays a role. We used substituted cysteine modification to address this question, a method fre-

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Section: Discussionmentioning

confidence: 94%

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Deeb¹,

Carland

Cooper

et al. 2007

Journal of Biological Chemistry

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“…Cysteine-scanning mutagenesis uses the highly reactive sulfhydryl moiety to determine the accessibility of side chains of amino acids. Two sulfhydryl-reactive reagents (36) that are often used in these studies are MTSET and MTSES, which are membrane-impermeant (37,38), react with porelining residues surrounded by water, and cannot reach residues within the hydrophobic bilayer.…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

Slepkov¹,

Rainey

Li³

et al. 2005

Journal of Biological Chemistry

150

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The Na ؉ /H ؉ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteineless NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe 161 3 Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD 3 OH:CDCl 3 :H 2 O mixture and in Me 2 SO. In CD 3 OH: CDCl 3 :H 2 O, TM IV was structured but not as a canonical ␣-helix. Residues Asp 159 -Leu 162 were a series of ␤-turns; residues Leu 165 -Pro 168 showed an extended structure, and residues Ile 169 -Phe 176 were helical in character. These three structured regions rotated quite freely with respect to the others. In Me 2 SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe 161 is a pore-lining residue.

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“…The substituted-cysteine-accessibility method (SCAM) is an approach to the characterization of channel structure (51)(52)(53) and binding-site structure (54)(55)(56) that probes the environment of any residue by mutating it to Cys and by characterizing the reaction of the Cys with sulfhydryl-specific reagents. Both because of the polarity of the methanethiosulfonates used (51,54) and because these reagents react at least 10 orders of magnitude faster with ionized thiolates than with unionized thiols (57), the reactions are directed to Cys at the water-accessible surface of the protein.…”

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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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Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates

Cited by 280 publications

References 50 publications

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

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

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