Functional Analysis of Transmembrane Domain 2 of the M1 Muscarinic Acetylcholine Receptor

Bee, Mark S.; Hulme, Edward C.

doi:10.1074/jbc.m703909200

Cited by 17 publications

(21 citation statements)

References 43 publications

(68 reference statements)

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“…For ACh, the present study is in good agreement with previously published work (Page et al, 1995;Lu and Hulme, 1999;Lu et al, 2001;Bee and Hulme, 2007;Goodwin et al, 2007). ACh stimulated all of the mutants with E Max values between 65% (Y404A) and 94% (C407A) of the value obtained for the wild-type receptor (values not significantly different to wild-type receptor; P Ͼ 0.1).…”

Section: Resultssupporting

confidence: 81%

“…Met79 and Tyr82 are better classified as belonging to the subsidiary pocket. This is consistent with the limited effects of the Y82A and M79A mutations on ACh-mediated activation of the M 1 mAChR (Lee et al, 1996;Bee and Hulme, 2007). Trp101 and Leu102 are in the "second shell" of the ACh binding site (Lu and Hulme, 1999).…”

supporting

confidence: 67%

“…This allowed three parameters to be estimated for each ligand: 1) the affinity of the ligand for the unoccupied receptor, measured by its pK d ; 2) its cooperativity with the tracer ligand NMS, measured by the cooperativity factor ␣; and 3) a slope factor, usually approximately 0.9. In addition, the experimental design provided a measure of the expression level of (Page et al, 1995;Lu and Hulme, 1999;Lu et al, 2001;Bee and Hulme, 2007).…”

Section: Resultsmentioning

confidence: 99%

“…With the exception of Y82A, the rat M 1 mutant receptors used in this work have been described previously (Page et al, 1995;Lu and Hulme, 1999;Lu et al, 2001;Bee and Hulme, 2007). They were cloned in the pCD expression vector and were transiently expressed in COS-7 cells grown in minimal essential medium-␣ supplemented with 10% newborn calf serum and 1% glutamate at 37°C in 5% CO 2 .…”

Section: Materials (ϫ)-N-[mentioning

confidence: 99%

“…Twenty-four hours after the transfection, cells were washed twice in warmed phosphate-buffered saline (PBS) and new minimal essential medium-␣ media added. When necessary, the media were supplemented with atropine (10 Ϫ6 M) to rescue the low-expressing mutant receptors (M79A, Y404A, and Y408A) as described previously (Lu et al, 2001;Bee and Hulme, 2007). Y82A was constructed by the QuikChange (Stratagene, La Jolla, CA) procedure and verified by dideoxy nucleotide sequencing.…”

Section: Materials (ϫ)-N-[mentioning

confidence: 99%

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Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors

Lebon¹,

Langmead²,

Tehan³

et al. 2008

Mol Pharmacol

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Point mutations and molecular modeling have been used to study the activation of the M 1 muscarinic acetylcholine receptor (mAChR) by the functionally selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them with N-desmethylclozapine (NDMC) and acetylcholine (ACh). Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although they were reduced by mutations of Tyr408. Signaling by AC-42, 77-LH-28-1, and NDMC was reduced by L102A and abolished by D105E, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105 to activate the M 1 mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by W101A, and their signaling activities were abolished by Y82A. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M 1 mAChR. We suggest the hypothesis that the side chain of Trp101 undergoes conformational isomerization, opening a novel binding site for the aromatic side chain of the AC-42 analogs. This may allow the positively charged piperidine nitrogen of the ligands to access the neighboring Asp105 carboxylate to activate signaling following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not seem to use this mechanism. Subtype-specific differences in the free energy of rotation of the side chain and indole ring of Trp101 might underlie the M 1 selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design.The M 1 muscarinic receptor (mAChR) is an attractive drug target to treat Alzheimer's disease and schizophrenia (Langmead et al., 2008b). The amino acid side chains that contact acetylcholine are modeled on the inward-facing segments of transmembrane (TM) helices 3, 4, 5, 6, and 7 within the outer leaflet of the lipid bilayer (Hulme et al., 2003). Their conservation between the five receptor subtypes has restricted the discovery of selective agonists.AC-42 is a novel ligand that activates M 1 mAChRs selectively (Spalding et al., 2002). Activation is little affected by alanine substitution of the orthosteric binding pocket residues Tyr381 (6.51; Ballesteros and Weinstein, 1995) and Asn382 (6.52) but requires sequences in the N terminus and TM1, extracellular loop 3, and the outer part of TM7. Thus AC-42 may activate M 1 mAChRs from a distinct "ectopic" site (Spalding et al., 2002). Supporting this, AC-42 can display allosteric interactions with N-methyl scopolamine (NMS) and atropine (Langmead et al., 2006). A study of TM3 showed that activation of M 1 mAChRs by AC-42 analogs was unaffected by alanine substitution of Tyr106 (3.33) neighboring the ACh counter-ion Asp105 (3.32) or Ser109 (3.36) one helical turn below but was inhibited by L102A (3.29) and paradoxically very ...

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Section: Resultssupporting

confidence: 81%

supporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Materials (ϫ)-N-[mentioning

confidence: 99%

Section: Materials (ϫ)-N-[mentioning

confidence: 99%

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Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors

Lebon¹,

Langmead²,

Tehan³

et al. 2008

Mol Pharmacol

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Structure–Function Studies of Muscarinic Acetylcholine Receptors

Leach

Simms

Sexton

et al. 2011

Muscarinic Receptors

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There has been great interest in the structure-function relationships of the muscarinic acetylcholine receptors (mAChRs) because these prototypical Family A/class 1 G protein-coupled receptors (GPCRs) are attractive therapeutic targets for both peripheral and central nervous system disorders. A multitude of drugs that act at the mAChRs have been identified over the years, but many of these show minimal selectivity for any one of the five mAChR subtypes over the others, which has hampered their development into therapeutics due to adverse side effects. The lack of drug specificity is primarily due to high sequence similarity in this family of receptor, especially in the orthosteric binding pocket. Thus, there remains an ongoing need for a molecular understanding of how mAChRs bind their ligands, and how selectivity in binding and activation can be achieved. Unfortunately, there remains a paucity of solved high-resolution structures of GPCRs, including the mAChRs, and thus most of our knowledge of structure-function mechanisms related to this receptor family to date has been obtained indirectly through approaches such as mutagenesis. Nonetheless, such studies have revealed a wealth of information that has led to novel insights and may be used to guide future rational drug design campaigns.

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G protein-coupled receptors of class A harness the energy of membrane potential to increase their sensitivity and selectivity

Shalaeva

Cherepanov

Galperin

et al. 2019

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The human genome contains about 700 genes of G protein-coupled receptors (GPCRs) of class A; these seven-helical membrane proteins are the targets of almost half of all known drugs. In the middle of the helix bundle, crystal structures revealed a highly conserved sodium-binding site, which is connected with the extracellular side by a water-filled tunnel. This binding site contains a sodium ion in GPCRs crystallized in their inactive conformations, but not in GPCRs that were trapped in agonist-bound active conformations. The escape route of the sodium ion upon the inactive-to-active transition and its very direction, either into the cytoplasm or back outside the cell, have remained obscure. By modeling the available experimental data we show that the sodium gradient over the cell membrane would increase the sensitivity of GPCRs if their activation were thermodynamically coupled to the sodium ion translocation into the cytoplasm, but decrease it if the sodium ion retreats into the extracellular space upon receptor activation. The model quantitatively describes the available data on both activation and suppression of distinct GPCRs by membrane voltage. The model also predicts selective amplification of the signal from (endogenous) agonists if only they, but not their (partial) analogs, could induce sodium translocation. Comparative structure and sequence analyses of sodium-binding GPCRs indicate a *

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Functional Analysis of Transmembrane Domain 2 of the M1 Muscarinic Acetylcholine Receptor

Abstract: Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M 1 muscarinic acetylcholine receptor, and of the highly conserved Asn 43 in TM1.

Cited by 17 publications

References 43 publications

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors

Structure–Function Studies of Muscarinic Acetylcholine Receptors

G protein-coupled receptors of class A harness the energy of membrane potential to increase their sensitivity and selectivity

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Functional Analysis of Transmembrane Domain 2 of the M1 Muscarinic Acetylcholine Receptor

Abstract: Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M 1 muscarinic acetylcholine receptor, and of the highly conserved Asn 43 in TM1.

Cited by 17 publications

References 43 publications

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M1 Muscarinic Acetylcholine Receptors

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M1 Muscarinic Acetylcholine Receptors

Structure–Function Studies of Muscarinic Acetylcholine Receptors

G protein-coupled receptors of class A harness the energy of membrane potential to increase their sensitivity and selectivity

Contact Info

Product

Resources

About

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors