M2 Pore Mutations Convert the Glycine Receptor Channel from Being Anion- to Cation-Selective

Keramidas, Angelo; Moorhouse, Andrew J.; French, Chris; Schofield, Peter R.; Barry, Peter H.

doi:10.1016/s0006-3495(00)76287-4

Cited by 112 publications

(129 citation statements)

References 39 publications

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“…In addition, it is likely that the inner and outer vestibules of the channel play a role in concentrating the appropriately charged ionic species (28); since we did not modify these regions, the 5-HT 3A -STM receptor may inadvertently be designed to effectively concentrate ions of the wrong charge. Indeed, similar observations reported for the converse glycine receptor STM described by Keramidas et al (2) make this an attractive hypothesis. We also investigated if D-tubocurarine or granisetron, both of which are antagonists of the 5-HT 3 receptor, could activate 5HT 3A -STM, but both compounds were inactive in this respect.…”

Section: Gating and Desensitization Of The 5-ht 3a -Stm-ligand-supporting

confidence: 75%

“…Although we have previously been able to use fluctuation noise analysis to study the unitary conductance of the WT and mutant 5-HT 3 receptors (19), preliminary studies on the 5-HT 3A -STM receptor failed to detect any resolvable channel noise (alternating current signal). 2 These findings may therefore reflect a reduced single channel conductance of the receptor, but further studies would be required to support this conclusion. It might, however, be anticipated that the conductance of the mutant receptor would be low, since we have mutated one of the rings of charged residues that Imoto et al (12) has shown affect conductance in the nACh receptor.…”

Section: Gating and Desensitization Of The 5-ht 3a -Stm-ligand-mentioning

confidence: 94%

“…Three mutations were found to be essential: the substitution of V13ЈT together with the neutralization of a ring of glutamate residues (EϪ1ЈA) and the adjacent insertion of the "extra" amino acid (proline) found in the anionic channels (referred to here as PϪ1Ј) in the M1-M2 loop. Subsequently, the "reverse" mutations in the ␣1 glycine receptor have been shown to change the ionic selectivity of this receptor (2). A schematic representation of these three rings of residues in the channel domain of the receptor is shown in Fig.…”

Section: -Htmentioning

confidence: 99%

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Conversion of the Ion Selectivity of the 5-HT3AReceptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily

Gunthorpe

Lummis

2001

Journal of Biological Chemistry

123

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The 5-hydroxytryptamine 3 (5-HT 3 ) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, ␥-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT 3A receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT 3A receptor from cationic to anionic; these were substitution of V13T in M2 together with neutralization of glutamate residues (E؊1A) and the adjacent insertion of a proline residue (P؊1) in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P Cl /P Na ‫؍‬ 12.3, P Na /P Cl ‫؍‬ 0.08), whereas WT receptors are predominantly permeable to sodium (P Na /P Cl > 20, P Cl /P Na < 0.05). Since the equivalent mutations have previously been shown to convert ␣7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500 -505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. 5-HT 31 receptors are ligand-gated ion channels (LGICs) whose activation results in membrane depolarization due to the gating of an integral cation-selective channel (3, 4). 5-HT 3 receptors can exist as homomeric assemblies of 5-HT 3A receptor subunits or heteromeric assemblies of 5-HT 3A and 5-HT 3B receptor subunits; these have distinct physical properties (5, 6). 5-HT 3 receptors form part of a phylogenetically linked superfamily of LGIC, which is typified by the nicotinic acetylcholine (nACh) receptor, also includes the glycine and ␥-aminobutyric acid type A receptors and is thought to have arisen from a common ancestor over 2000 million years ago (7). All of these receptors are believed to be constructed from a pseudopentameric arrangement of subunits around a central water-filled pore. Individual subunits are predicted to contain a large Nterminal domain and four transmembrane spanning domains (M1-M4), arranged such that M2 (see Fig. 1 for residue numbering and lettering in MZ) delineates the wall of the channel. Agonist binding to the N-terminal domain results in a conformational change that is communicated to the pore domain, resulting in the opening of the channel. Evidence suggests that as well as the conservation of structural features, the underlying molecular mechanisms of operation of these receptors are also conserved (8,9). However, once the channels have opened, one crucial difference becomes clear; whereas the integral ion channels of the 5-HT 3 a...

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Section: Gating and Desensitization Of The 5-ht 3a -Stm-ligand-supporting

confidence: 75%

Section: Gating and Desensitization Of The 5-ht 3a -Stm-ligand-mentioning

confidence: 94%

Section: -Htmentioning

confidence: 99%

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Conversion of the Ion Selectivity of the 5-HT3AReceptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily

Gunthorpe

Lummis

2001

Journal of Biological Chemistry

123

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“…The engineered cysteines are located in the M1-M2 loop (G-2ЈC) and on the cytoplasmic side of the M2 helix (S2ЈC), a region known to be important for permeability and ion selectivity (15)(16)(17)(18)(19)(20)(21)(22)(23). Because of the magnitude and persistence of inhibition in both oocyte and HEK-293T cell expression systems, we investigated in more detail the Cd 2ϩ inhibition of the S2ЈC receptor.…”

Section: Discussionmentioning

confidence: 99%

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Panicker

Cruz

Arrabit

et al. 2004

Journal of Biological Chemistry

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Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT 3A ) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd 2؉ ) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2 C and, to a lesser extent, G-2 C, showed high affinity inhibition with Cd 2؉ Ligand-gated ion channel (LGIC) 1 receptors are responsible for rapid chemical transmission between neurons in the nervous system (1). Alterations in the response of ligand-gated receptors have profound effects on neuronal activity in the brain. For example, mutations in nicotinic acetylcholine-gated receptors (nAChRs) and glycine-gated receptors have been linked to certain forms of epilepsy and startle disease, respectively (2-4).LGIC receptors that respond to acetylcholine, ␥-aminobutyric acid, glycine, or serotonin (5-HT) possess a conserved pair of extracellular cysteines in the N-terminal domain ("Cys loop" receptors) and are composed of five subunits, each containing four putative transmembrane domains (5). Of these four domains, the second transmembrane domain (M2) lines the majority of the water-filled pore (Fig. 1A).The rapid response of LGIC receptors is achieved by the energetic coupling of agonist binding in the extracellular Nterminal domain with a "gate" located 60 Å away in the receptor pore (M2 domain) (6). Several studies with LGIC receptors suggest that the gate is situated in the middle of the M2 (6 -12) (but see Ref. 13). The primary determinants of selectivity appear to be located below the gate, near the cytoplasmic membrane surface. Mutations in the cytoplasmic end of the M2 alter ion selectivity (14 -17), and several recent studies show that by mutating sites in this region, cation-conducting LGIC can be converted to anion-conducting channels and vice versa (18 -23). Because permeability of like-charged molecules in LGIC appears to be largely dependent on size, it is believed that the narrowest region of the open LGIC pore coincides with the channel selectivity filter, which is formed by a portion of the M1-M2 loop and the cytoplasmic side of the M2 helix (16,24). Consistent with this region being narrow, site-specific mutations in the filter alter conductivity in a manner that depends upon side-chain volume (8,24). Thus, the pore of the open receptor can be envisaged as a funnel, with a wide extracellular vestibule that tapers to a constriction at the cytoplasmic side of the membrane, where ion selectivity is determined (25).What is known about the extent of movement in the cytoplasmic selectivity filter? The 9-Å three-dimensional structures of open and closed Torpedo nAChRs derived fro...

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“…How such functional distinctions arise among channels with highly conserved features has been a long-standing question. Previous work has focused on some specific charged loci in one or a few pLGIC members to identify potential influences on charge selectivity (5)(6)(7)(8). However, as noted in a paper by Cymes and Grosman in PNAS (9), a detailed analysis of the literature reveals that a large number of discrepancies exist and that no consensus mechanism has yet been attained.…”

mentioning

confidence: 99%

Engineering differential charge selectivity from a single structural template

Zhou¹,

Lingle²

2016

Proc. Natl. Acad. Sci. U.S.A.

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For any ion channel, both the identity and fundamental physiological roles are largely defined by ion selectivity. It is the regulated passage of some ions, but not others, across a cell membrane that determines specific membrane potential (V m ) changes that occur with channel activation. For example, opening of potassium (K + )-selective channels typically drives V m to negative values, generally reducing excitability, whereas activation of voltagedependent sodium (Na + )-or calcium (Ca 2+ )-selective channels contributes to strong depolarization. For the superfamily of tetrameric, strongly cation-selective channels, the determinants of selectivity arise in part from favored occupancy by permeant ions at one or more specific sites within the permeation pathway, sites readily identified in available structures (1, 2). Specific coordination of permeant cations by carbonyl oxygens or carboxylic acid side chains favors occupancy by the permeating species over other cations of slightly different radii (3, 4). However, other ion channels are not so explicitly selective and have evolved alternative filtering strategies. A remarkable case is that of members of the pentameric ligand-gated ion channel (pLGIC) superfamily, in which each member shares similar structural features, but some are cation-selective whereas others are anion-selective. How such functional distinctions arise among channels with highly conserved features has been a long-standing question. Previous work has focused on some specific charged loci in one or a few pLGIC members to identify potential influences on charge selectivity (5-8). However, as noted in a paper by Cymes and Grosman in PNAS (9), a detailed analysis of the literature reveals that a large number of discrepancies exist and that no consensus mechanism has yet been attained. For example, the elementary question as to whether charge selectivity is governed by interactions of the permeating ions with backbone atoms or side-chain atoms had not been answered conclusivelyfor the pLGIC superfamily-before this work.Taking an approach that is really one of classic comparative physiology, but applied at the molecular level, Cymes and Grosman (9) undertake an exhaustive mutational analysis of various elements that might influence charge selectivity, defining K + /Cl − permeability ratios using a traditional dilution-condition evaluation of ion selectivity. They compare six distinct pLGIC templates, including four cation-selective channels [the adult-muscle (α1) 2 βδe nicotinic acetylcholine receptor (nAChR), the homomeric serotonin type 3A receptor (5-HT 3A R), the heteromeric serotonin type 3A-3B receptor (5-HT 3A-3B R) from mammals, and the bacterial ELIC channel] and two anion-selective channels [the α1 glycine receptor (α1GlyR) from mammals , and 0′ are rendered as ball-and-chain in white, blue, and red, respectively. The ion permeation pathway is denoted by the central Na + (orange sphere in the GLIC structure). B3 shows a zoom-in view of these three residues in a GLIC M2, with homologous re...

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M2 Pore Mutations Convert the Glycine Receptor Channel from Being Anion- to Cation-Selective

Cited by 112 publications

References 39 publications

Conversion of the Ion Selectivity of the 5-HT3AReceptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily

Conversion of the Ion Selectivity of the 5-HT3AReceptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Engineering differential charge selectivity from a single structural template

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