Cys-loop Research Articles

Barbiturates Require the N Terminus and First Transmembrane Domain of the δ Subunit for Enhancement of α1β3δ GABAA Receptor Currents

GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.

Pharmacological Characterization of the Allosteric Modulator Desformylflustrabromine and Its Interaction with α4β2 Neuronal Nicotinic Acetylcholine Receptor Orthosteric Ligands

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. nAChRs are involved in modulating nicotinic-based signal transmission in the central nervous system and are implicated in a range of disorders. Desformylflustrabromine (dFBr) is a positive allosteric modulator that potentiates alpha4beta2 nAChRs. It has been reported that dFBr is selective for the alpha4beta2 receptor relative to other common nAChR subtypes (Neurosci Lett 373:144-149, 2005). Coapplication of dFBr with acetylcholine (ACh) produces a bell-shaped dose-response curve with a peak potentiation of more than 265% (Bioorg Med Chem Lett 17:4855-4860, 2007) at dFBr concentrations <10 microM and inhibition of responses at concentrations >10 microM. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Currents to both partial and full agonists were potentiated by dFBr. Responses to low-efficacy agonists were potentiated significantly more than responses to high-efficacy agonists. Antagonist pIC(50) values were unaffected by coapplication of dFBr. In addition to its potentiating effects, dFBr was able to induce current spikes when applied to desensitized receptors, suggestive of a shift in equilibrium from the desensitized to open conformation. In contrast to potentiation, inhibition of ACh responses by dFBr depends on membrane potential and is probably the result of open-channel block by dFBr and ACh. Our data indicate distinct mechanisms for the potentiation and inhibition components of dFBr action. dFBr could prove useful for therapeutic enhancement of responses at alpha4beta2-containing synapses.

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Proper regulation of neurotransmission requires that ligand-activated ion channels remain closed until agonist binds. How channels then open remains poorly understood. Glycine receptor (GlyR) gating is initiated by agonist binding at interfaces between adjacent subunits in the extracellular domain. Aspartate-97, located at the alpha1 GlyR interface, is a conserved residue in the cys-loop receptor superfamily. The mutation of D97 to arginine (D97R) causes spontaneous channel opening, with open and closed dwell times similar to those of maximally activated WT GlyR. Using a model of the N-terminal domain of the alpha1 GlyR, we hypothesized that an arginine-119 residue was forming intersubunit electrostatic bonds with D97. The D97R/R119E charge reversal restored this interaction, stabilizing channels in their closed states. Cysteine substitution shows that this link occurs between adjacent subunits. This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation.

In silico point mutation and evolutionary trace analysis applied to nicotinic acetylcholine receptors in deciphering ligand-binding surfaces

The nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily and contain ligand gated ion channels (LGIC). These receptors are located mostly in the central nervous system (CNS) and peripheral nervous system (PNS). nAChRs reside at pre-synaptic regions to mediate acetylcholine neurotransmission and in the post synaptic membrane to propagate nerve impulses through neurons via acetylcholine. Malfunction of this neurotransmitter receptor is believed to cause various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and schizophrenia, and nAChRs are thus important drug targets. In the present work, starting from an earlier model of pentameric alpha7nAChR, a considerable effort has been taken to investigate interaction with ligands by performing docking studies with a diverse array of agonists and antagonists. Analysis of these docking complexes reveals identification of possible ligand-interacting residues. Some of these residues, e.g. Ser34, Gln55, Ser146, and Tyr166, which are evolutionarily conserved, were specifically subjected to virtual mutations based on their amino acid properties and found to be highly sensitive in the presence of antagonists by docking. Further, the study was extended using evolutionary trace analysis, revealing conserved and class-specific residues close to the putative ligand-binding site, further supporting the results of docking experiments.

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and gamma-aminobutyric acid type A receptors (GlyRs and GABA(A)Rs, respectively) in the fluorescence-based FLIPR(TM) Membrane Potential assay. The compound inhibited the signaling of all GABA(A)R subtypes included in the study with high nanomolar/low micromolar IC(50) values, except the rho 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC(50) values at the homomeric alpha1 and alpha2 GlyRs, whereas it was inactive at the heteromeric alpha 1 beta and alpha 2 beta subtypes at concentrations up to 300 microm. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6' M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA(A)R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a alpha1 GlyR homology model revealed two distinct putative binding sites formed by the 6' M2 residues together with the 2' residues and the 10' and 13' residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor.

Identification and Functional Characterization of a Novel Acetylcholine-Binding Protein from the Marine Annelid Capitella teleta

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.

Advances and hold-ups in the study of structure, function and regulation of Cys-loop ligand-gated ion channels and receptors

The Cys-loop receptor channels, including the nicotinic acetylcholine (nAChR), serotonin 5-HT3, GABAA, GABAC and glycine receptor channels, are widely expressed in the nervous system (both central and peripheral) where they regulate excitability and neurotransmitter release, as well as at the endplate where they induce neuromuscular contraction. Furthermore dysfunctions in these receptors are thought to be involved in a variety of diseases and disorders, including (but not limited to) Alzheimer's disease, Parkinson's disease, epilepsy, schizophrenia, myasthenic syndromes, depression and substance abuse. Presently the Cys-loop receptors serve as molecular targets for a variety of clinically important drugs, such as muscle relaxants, tranquillizers, anticonvulsants and anti-emetics. Thus, these receptors are presently critical targets for the development of therapeutics to treat a variety of neurological diseases and disorders.

Gating of nicotinic ACh receptors: latest insights into ligand binding and function

Nicotinic acetylcholine receptors (nAChRs) are in the superfamily of cys-loop receptors, and are widely expressed in the nervous system where they participate in a variety of physiological functions, including regulating excitability and neurotransmitter release, as well as neuromuscular contraction. Members of the cys-loop family of receptors, which also includes the molluscan ACh-binding protein (AChBP), a soluble protein that is analogous to the extracellular ligand-binding domain of the cys-loop receptors, are pentameric assemblies of five subunits, with each subunit arranged around a central pore. The binding of ACh to the extracellular interface between two subunits induces channel opening. With the recent 4 A resolution of the Torpedo nAChR, and the crystal structure of the AChBP, much has been learned about the structure of the ligand-binding domain and the channel pore, as well as major structural rearrangements that may confer channel opening, including a major rearrangement of the C-loop within the ligand binding pocket, and perhaps other regions including the F-loop (the beta8-beta9 linker), the beta1-beta2 linker and the cys-loop. Here I will review the latest findings from my lab aimed at a further understanding of the function of the neuronal nAChR channels (and in particular the role of desensitization), and our search for novel AChBP species that may lead to a further understanding of the function of the cys-loop receptor family.

A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3receptor function

The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT(3)) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT(3)B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.

Multifunctional Basic Motif in the Glycine Receptor Intracellular Domain Induces Subunit-specific Sorting

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.

Chemical Scale Studies of the Phe-Pro Conserved Motif in the Cys Loop of Cys Loop Receptors

The functions of two conserved residues, Phe(135) and Pro(136), located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe(135), backbone conformation at Pro(136), side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function.

Microsecond Simulations Show that Ethanol Binds between Subunits and Stabilizes the Open Form of a Glycine Receptor Model

Cys-loop receptors are a superfamily of ligand gated ion channels comprising such receptors as acetylcholine, serotonin, Glycine and GABA. Despite the low level of sequence conservation all these receptors are thought to share common structural characteristics. Numerous experiments describe in detail the pore channel and the extracellular domain. However due to the low sequence conservation between members of Cys-loop family, and the lack of any high resolution structures of these receptors in the trans-membrane domain (TMD), little information is available concerning the potential allosteric binding site. This potentiating binding site is of extreme importance for a pharmacological perspective, as it is the target of anesthetic an alcohol molecules.Recently the crystal structure of the open form of Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) has been solved [1]. As Glycine receptors (GlyR) are the closest receptors in terms of sequence, to GLIC, we decided to create a homology model of the human homomeric alpha-1 GlyR. Furthermore we chose the well-known ethanol molecule as a ligand target for studying the potentiating effect of such molecules on Cys-loop receptors [2]. In total we present 2 microseconds of molecular dynamics simulations, the longest simulation ever presented for Cys-loop receptors. Our simulations show a spontaneous binding of ethanol in cavities of the TMD. These cavities are located between subunits of GlyR TMD, and involve several residues previously identified by mutations as crucial for the potentiating effect of GlyR by ethanol. We also show that ethanol is stabilizing the open form of the GlyR, which could explain the effect of allosteric ligand on Cys-loop receptors.[1] Bocquet, N, et al. Nature (2009): (457)111-114.[2] Harris, RA, Trudell, JR and Mihic, SJ. Sci Signal. (2008): 1(28):re7.

Energetics of Allosteric ion Binding to a Ligand-Gated ion Channel

Allosteric regulation of ligand-gated ion channels (LGICs) is ubiquitous, involving discrete transitions between resting, conducting, and desensitized states, driven by agonist binding. Small molecules that bind at specific sites have also been reported to act as allosteric modulators in many LGICs, such as glutamate, P2X, and Cys-loop receptors, but an understanding of the underlying molecular mechanisms is sparse. Of these, perhaps the best studied are kainate subtype glutamate receptors which require both extracellular Na+ and Cl- for receptor function. Studies on glutamate receptors (iGluRs) have defined the ligand binding domain (LBD) dimer assembly as the key functional unit that controls channel activation and desensitization. Using crystallographic and electrophysiological approaches, we have previously shown that for kainate, but not AMPA iGluRs, the binding of Na+ and Cl- ions to discrete, electrostatically coupled sites in the extracellular LBD dimer regulates the rate of entry into the desensitized state, which occurs when the dimer interface ruptures and the channel closes. We have now dissected the energetic effects of allosteric ions on kainate receptor dimer stability in solution using analytical ultracentrifugation. Our results show that Na+ and Cl- ions modulate dimer affinity as much as 50-fold, and that removal of either ion disrupts the dimer. We further tested the generality of this model of ion action for Ca2+ modulation of the orphan iGluR delta2 that crystallizes as a dimer which binds Ca2+. Our results indicate that ions can contribute substantial free energy to active state stabilization in both these receptors, and we postulate that in contrast to AMPARs, the dimer interface in these receptors may be intrinsically weak to serve a functional role: Allowing ion modulation. Our results provide quantitative measurements of the energetic effects of allosteric ion binding on a LGIC.

M2 Segment Accessibility in The Prokaryotic Proton-Gated Cys-loop Receptor Channel from Gloeobacter Violaceus in Closed and Open States

The prokaryotic homopentameric proton-gated cation channel from Gloeobacter violaceus (Glic) is a putative homologue of the Cys-loop family of neurotransmitter-gated channels, with an ∼200 residue N-terminal extracellular domain and 4 transmembrane α-helices per subunit. Glic lacks the large cytoplasmic loop between M3 and M4 and the signature disulfide linkage. High resolution crystal structures of Glic were recently published. To determine whether the Glic structure is a good model for eukaryotic Cys-loop receptors, we tested the accessibility of 30 individual cysteine substitutions in M2 and the M2-M3 loop of Glic to p-chloromercuribenzenesulfonate (pCMBS-) in the closed state (pH 7.5) or in a sub-maximally activated state (pH 5). In 11 of the 30 Cys mutants the proton-induced currents were not significantly different than those in water-injected oocytes. Of the mutants tested from −2′ to 7′, E221C (−2′) and T225C (2′) were reactive at both pH 7.5 and 5.0, V224C (1′) and T230C (7′) were reactive only at pH 5.0, and A222C (−1′) was reactive only at pH 7.5. Reactions occurred at rates <100 M−1s−1. The 8′ and 9′ mutants showed aberrant gating properties. From 10′ to 27′ pCMBS- reacted with all residues except H234C (11′), V241C (18′), T243C (20′) and L245C (22′). Reaction rates at residues more extracellular than 13′ were >1000 M−1s−1. While a clear α-helical pattern is not apparent, given recently published X-ray crystallographic data we infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is loosely packed from 10′ to the extracellular end. The chemical accessibility data suggest a more loosely packed structure than the crystal structures, which may represent a desensitized state.

Multiple Modes of Interaction between the Nicotinic Acetylcholine Receptor and Isoflurane Observed Through Long Time Simulations

Understanding of the molecular mechanisms through which anesthetics affect function of ligand gated ion channels (LGIC) like the nicotinic acetylcholine receptor (nAChR) is complicated by the multitude of binding sites and possible mechanisms that are consistent with experimental data, including pore-block, competitive binding, and allosteric mechanisms. Molecular dynamics (MD) studies of general anesthetics and Cys-loop receptors have been hindered by low resolution and unstable structures and the computational cost of simulating large systems for the long times required to observe all binding modes. We present long time (400 ns) simulations in which isoflurane introduced into the water surrounding the nAChR proceeded to bind to four main classes of site on the protein (as well as partition into the membrane) : 1) the hydrophobic constriction of the channel lumen, effectively blocking the pore 2) the agonist site 3) the interface between subunits, in the transmembrane domain, 4) in the center of some subunits, in the transmembrane domain on the intracellular half. Sites 1) and 2) are most likely inhibitory sites that may not be present in anionic cys-loop channels (which are potentiated by anesthetics);we find analogous sites in a 200 ns simulation of the prokaryotic cationic LGIC from Gloeobacter violaceus (for which two high resolution structures are available). Isoflurane bound to site 3) is found to have distinct and statistically significant effects on the structure and dynamics of the adjacent critical M2-M3 loop, with repercussions in the pore-lining M2 helices,consistent with an allosteric potentiating mechanism that may account for the reverse effect of isoflurane on the GABA and glycine receptors.

Aromatic Interfaces between Transmembrane Helices M1/M4 and M3/M4 Play a Key Role in Cys-loop Receptor Assembly

Cys-loop receptors, also designated pentameric ligand-gated ion channels (pLGICs) include nicotinic acetylcholine receptors (nAChRs), serotonin type 3 receptors (5HT3Rs), γ-amino butyric acid type-A receptors (GABAARs) and glycine receptors (GlyRs). pLGICs function as obligate pentamers linked by non-covalent interactions between the N-terminal extracellular domains of identical or homologous subunits. Here we show that expression of GlyR α1 or 5HT3A subunits in two separate fragments (one containing the ectodomain and transmembrane domains M1-M3, and the other the fourth transmembrane domain M4) results in the assembly of functional pLGICs indistinguishable in their electrophysiological properties from wt pLGICs assembled from contiguous subunits. Alanine scanning of M1, M3 and M4 of the GlyR α1 subunit identified a total of 12 aromatic residues as important or crucial for pentameric assembly. The assembly-relevant aromatic residues cluster in one face of each helix. Homology modelling based on crystal structures (Hilf & Dutzler 2008; Bocquet et al 2008) predicted π- π interactions between the aromatic face of the M4 helix and three or two aromatic residues located in the M1 helix (Tyr228, Trp239, and Phe242) and the M3 helix (Trp286, Phe293), respectively. The loss of homopentamer formation and function seen upon alanine replacement of any of these contact residues strongly supports the existence of a membrane-embedded network of pairwisely interacting aromatic side chains that compacts and stabilizes the membrane core region of the GlyR. We infer from these results that a precise geometric arrangement of transmembrane helices defined by the tri-helical aromatic network is a prerequisite to allow the circular arrangement of the subunits stabilized essentially by earlier occurring random subunit interactions between the ectodomains.

Molecular Dynamics Simulation Reveals a Possible Mechanism of Activation of the Prokaryotic Proton Activated Channel Glic

Recent bioinformatics and high resolution structural determination revealed prokaryotic ancestors to the superfamily of eukaryotic Cys-loop receptors. One of these channels, called GLIC, is proton-activated and may provide advantages to investigating mechanisms underlying activation of Cys-loop receptors. Here, we used all-atom molecular dynamics simulations to uncover a possible mechanism of activation of the GLIC channel. We started with the x-ray structure of GLIC in the open state and subjected it to simulation under normal conditions. During the 10 ns simulation the channel appeared to close. Then we screened the apparent closed structure for the presence of salt-bridges, and compared the result to salt bridges in the original open x-ray structure. We found 11 salt bridges per subunit that were not present in the open structure. We then protonated the corresponding 11 acidic residues, conducted another 10 ns simulation and found that the channel remained open. We then extracted the final structural frames from the two MD simulations and employed single channel current simulations using the coarse-grained Biology Monte Carlo method. The open structure resulted in a significant ionic current, whereas the closed structure exhibited none. Thus, the 11 acidic residues emerge as likely candidates for mediating proton activation of the GLIC channel.

Molecular Determinants in Glycine and GABA-A Receptors that Sense Intracellular Chloride Concentration

GABA-A and glycine receptors are ligand-gated ion channels belonging to the Cys-loop superfamily. They are permeable to anions and mediate fast synaptic inhibition in the central nervous system. We recently showed that intracellular chloride can affect the time course of deactivation of these channels (1). The aim of the present work was to identify the residues that are involved in “sensing” the intracellular chloride concentration.We used the fast concentration jump technique to apply brief pulses (1-3 ms) of saturating glycine or GABA to outside-out patches from transiently-transfected HEK cells expressing alpha1 or alpha1 beta glycine receptors or alpha1 beta2 gamma2L GABA-A receptors. The effect of intracellular chloride could be mediated either by residues in intracellular loops, such as M1-M2 and M3-M4, or by the residues lining the channel itself. We used a combination of deletion and Ala/Cys-scanning mutagenesis approaches to address this question.In other nicotinic channels, the long M3-M4 intracellular loop can be replaced by the equivalent domain (7 amino acids) of the orthologous prokaryotic channel from Gloeobacter violaceus without losing channel function (2). We found that chimeric constructs of glycine and GABA-A receptors containing the prokaryotic M3-M4 domain retained modulation by intracellular chloride, suggesting that the major cytoplasmic domain is unlikely to mediate this effect. The results of mutagenesis of residues facing the ion permeation pathway on the contrary strongly suggests that chloride ions affect channel kinetic by binding to a site along the pore; we have identified conserved residues that reduce the differences between the deactivation rates measured at different chloride concentrations.(1) Pitt, Sivilotti and Beato (2008) J.Neurosci 28, 11454-11467(2) Jansen, Bali and Akabas (2008) J.Gen.Physiol. 131, 137-146

Length and Composition of the 5HT3A Receptor M3M4 Loop Effects Channel Expression and Desensitization

5HT3A receptors are members of the Cys-loop receptor family of neurotransmitter-gated ion channels. These proteins share a similar structure; each subunit contains an amino-terminal extracellular domain, four transmembrane (TM) domains and a large intracellular loop (∼100 residues) between TM3 and TM4. Prokaryotic Cys-loop homologues lack a large intracellular loop, having instead a loop of less than 15 residues. To date, very few studies have looked at the effects of the M3M4 loop on channel function, kinetics and desensitization. To investigate the role of the M3M4 loop in receptor function we replaced the 5-HT3A M3M4 loop with chains of alanine repeats (5-HT3A-An=1-7) and studied the electrophysiological and biochemical properties of the resulting homomeric channels. All mutants were functional with 5-HT EC50's similar to wild type receptors; however desensitization times differed greatly amongst the mutants. 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, or 5-HT3A-A7 demonstrated channel desensitization rates similar to wild type 5-HT3A channels; however 5-HT3A-A1, 5-HT3A-A3, or5-HT3A-A5 had desensitization rates an order of magnitude faster than wildtype. Additionally, the 5-HT3A-A1 construct enters a non-binding, non-functional state after initial opening, from which it cannot recover. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function; however the length of the loop and its amino acid composition can have significant effects on channel kinetics and desensitization rate. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments in the channel protein as it undergoes gating-induced conformational changes. Thus, while the M3M4 loop is not essential for channel assembly and function it can exert significant effects on the channel kinetics.

An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.