Ligand-gated Ion Channel Superfamily Research Articles

Association of the nicotinic receptor beta 2 subunit and febrile seizures

The nicotinic acetylcholine receptors are members of a superfamily of ligand-gated ion channels that mediate fast signal transmission at synapses. Mutations in neuronal nicotinic acetylcholine receptor beta 2 subunit have been associated with autosomal dominant nocturnal frontal lobe epilepsies. A major challenge is to establish whether the monogenic epilepsy gene also contributes to the common epilepsies. Because febrile seizures represent the majority of childhood seizures, and a genetic predisposition, we investigated the possibility that the nicotinic acetylcholine receptor beta 2 subunit might be involved in the etiology of febrile seizures. Children were divided into two groups: those with febrile seizures (group 1; n = 104) and control patients (group 2; n = 83). Polymerase chain reaction was used to identify the G/C and T/C polymorphisms of the nicotinic acetylcholine receptor beta 2 subunit gene, which is mapped on chromosome 1. Genotypes and allelic frequencies for nicotinic acetylcholine receptor beta 2 subunit gene polymorphisms in both groups were compared. The results indicated that genotypes and allelic frequencies in both groups were not significantly different. These data suggest that nicotinic acetylcholine receptor beta 2 subunit polymorphisms are not a useful marker for prediction of the susceptibility to febrile seizures.

Pharmacological Survey of Medicinal Plants for Activity at Ligand-Gated Ion Channels: Selective Interaction with 5-HT3 Receptors

The ligand-gated anion-selective ion channels are part of a superfamily of ligand-gated ion channels (LGICs) that are responsible for a majority of inhibitory signaling in the central nervous system and are the targets of numerous therapeutics. Aqueous, organic and alcoholic extracts of 47 Chinese, Bolivian and Pakistani medicinal plants were evaluated for the ability to modulate the activity of GABAA receptors. Extracts were initially screened for their ability to modulate activity of α1β2γ2 GABAA receptors expressed in HEK 293 cells. Based on the initial screen, two extracts derived from members of the Asteraceae family, Xanthium spinosum and Senecio mathewsii, were chosen for more detailed analysis. Xanthium spinosum inhibited GABAA receptor function, with an IC50 of 50 ± 10 µg/ml, while Senecio mathewsii inhibited GABAA receptor activity with an IC50 of 35 ± 3.0 µg/ml. To assess the selectivity of interaction, these extracts were also tested on two other members of the LGIC superfamily a) glycine receptors, a distinct inhibitory neurotransmitter receptor and b) 5-HT3A receptors, a cation-selective receptor. At a concentration of Xanthium spinosum that blocked 70% of GABA-activated current, only 15% of glycine-gated current, recorded from recombinant α1 glycine receptors, was blocked, suggesting a lower affinity of Xanthium spinosum for glycine receptors. Senecio mathewsii had larger inhibitory effects on glycine receptors than Xanthium spinosum, although the apparent affinity still was estimated to be three-fold lower than that seen for GABAA receptors. Xanthium spinosum and Senecio mathewsii also inhibited 5-HT3A receptor functions. Notably, the IC50 of both extracts was seven to ten-fold lower than that observed for GABAA receptors. Thus, the rank order of potency for organic extracts from both Xanthium spinosum and Senecio mathewsii was 5-HT3A receptors > GABAA receptors > glycine receptors. Therefore, these extracts may be a source of novel compounds that may serve as lead molecules for the development of novel 5-HT3 receptor antagonists.

Molecular Determinants of Proton Modulation of Glycine Receptors

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.

Terpene Trilactones from Ginkgo biloba Are Antagonists of Cortical Glycine and GABAA Receptors

Glycine and gamma-aminobutyric acid, type A (GABA(A)) receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory synaptic transmission in the adult central nervous system. During development, the activation of these receptors leads to membrane depolarization. Ligands for the two receptors have important implications both in disease therapy and as pharmacological tools. Terpene trilactones (ginkgolides and bilobalide) are unique constituents of Ginkgo biloba extracts that have various effects on the central nervous system. We have investigated the relative potency of these compounds on glycine and GABA(A) receptors. We find that most of the ginkgolides are selective and potent antagonists of the glycine receptor. Bilobalide, the single major component in G. biloba extracts, also reduces glycine-induced currents, although to a lesser extent. Both ginkgolides and bilobalide inhibit GABA(A) receptors, with bilobalide demonstrating a more potent effect. Additionally, we provide evidence that open channels are required for glycine receptor inhibition by ginkgolides. Finally, we employ molecular modeling to elucidate the similarities and differences in the structure of the terpene trilactones to account for the pharmacological properties of these compounds and demonstrate a striking similarity between ginkgolides and picrotoxinin, a GABA(A) and recombinant glycine alpha-homomeric receptor antagonist.

Structure and function of AChBP, homologue of the ligand-binding domain of the nicotinic acetylcholine receptor.

Acetylcholine-binding protein (AChBP) is a novel protein with high similarity to the extracellular domain of the nicotinic acetylcholine receptor. AChBP lacks the transmembrane domains and intracellular loops typical for the nAChRs. AChBP is secreted from glia cells in the central nervous system of the freshwater snail, Lymnaea stagnalis, where it modulates synaptic transmission. AChBP forms homopentamers with pharmacology that resembles the alpha(7)-type of nicotinic receptors. As such, AChBP is a good model for the ligand-binding domain of the nAChRs. In the crystal structure of AChBP at 2.7 A, each protomer has a modified immunoglobulin fold. Almost all residues previously shown to be involved in ligand binding in the nicotinic receptor are found in a pocket at the subunit interface, which is lined with aromatic residues. The AChBP crystal structure explains many of the biochemical studies on the nicotinic acetylcholine receptors. Surprisingly, the interface between protomers is relatively weakly conserved between families in the superfamily of pentameric ligand-gated ion channels. The lack of conservation has implications for the mechanism of gating of the ion channels.

Comparative modeling of GABA A receptors: limits, insights, future developments

GABA A receptors are chloride ion channels that mediate fast synaptic transmission and belong to a superfamily of pentameric ligand-gated ion channels. The recently published crystal structure of the acetylcholine binding protein can be used as a template for comparative modeling of the extracellular domain of GABA A receptors. In this commentary, difficulties with comparative modeling at low sequence identity are discussed, the degree of structural conservation to be expected within the superfamily is analyzed and numerical estimates of model uncertainties in functional regions are provided. Topography of the binding sites at subunit-interfaces is examined and possible targets for rational mutagenesis studies are suggested. Allosteric motions are considered and a mechanism for mediation of positive cooperativity at the benzodiazepine site is proposed.

A Basic Cluster Determines Topology of the Cytoplasmic M3-M4 Loop of the Glycine Receptor α1 Subunit

The inhibitory glycine receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors, which are composed of homologous subunits with four transmembrane segments (M1-M4), each. Here, we demonstrate that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop (designated M3-M4 loop) following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment.

Prediction of 5-HT 3 Receptor Agonist-Binding Residues Using Homology Modeling

5-HT 3 receptors demonstrate significant structural and functional homology to other members of the Cys-loop ligand-gated ion channel superfamily. The extracellular domains of these receptors share similar sequence homology (∼20%) with Limnaea acetylcholine binding protein, for which an x-ray crystal structure is available. We used this structure as a template for computer-based homology modeling of the 5-HT 3 receptor extracellular domain. AutoDock software was used to dock 5-HT into the putative 5-HT 3 receptor ligand-binding site, resulting in seven alternative energetically favorable models. Residues located no more than 5 Å from the docked 5-HT were identified for each model; of these, 12 were found to be common to all seven models with five others present in only certain models. Some docking models reflected the cation- π interaction previously demonstrated for W183, and data from these and other studies were used to define our preferred models.

The GABA A receptor antagonist picrotoxin inhibits 5-hydroxytryptamine type 3A receptors

For a number of years it has been known that the CNS convulsant picrotoxin inhibits the GABA A receptor, an anion-selective member of the ligand-gated ion channel (LGIC) superfamily. PTX also inhibits other anion-selective LGIC members, such as GABA C, glycine and glutamate-gated Cl - channels. In the present report, we tested the ability of picrotoxin to inhibit cation-selective 5-HT 3A receptors. Murine 5-HT 3A receptors were expressed in HEK293 cells, and functionally evaluated using whole-cell patch clamp recording. Picrotoxin inhibited 5-HT-gated currents in a concentration-dependent manner, with an IC 50 of approximately 30 μM. Moreover, the blockade by PTX was non-competitive and use-facilitated. Pentylenetetrazole and U-93631, ligands that act at a domain similar to that of picrotoxin in GABA A receptors, also inhibited the 5-HT 3A receptor. For each ligand tested, its potency was 5–10 fold lower than typically observed in GABA A receptors. Our results demonstrate that, in addition to being a relatively non-selective inhibitor of anionic LGICs, picrotoxin also inhibits the cation-selective 5-HT 3A receptor. Moreover, the fact that both PTZ and U-93631 similarly inhibit the 5-HT 3A receptor is consistent with the suggestion that the site of picrotoxin action in this receptor may be comparable to that in anion-selective LGICs.

Coupling of agonist binding to channel gating in the GABA(A) receptor.

Neurotransmitters such as acetylcholine and GABA (gamma-aminobutyric acid) mediate rapid synaptic transmission by activating receptors belonging to the gene superfamily of ligand-gated ion channels (LGICs). These channels are pentameric proteins that function as signal transducers, converting chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with a ligand-binding site, triggering a conformational change in the protein that results in the opening of an ion channel. This process, which is known as 'gating', occurs rapidly and reversibly, but the molecular rearrangements involved are not well understood. Here we show that optimal gating in the GABA(A) receptor, a member of the LGIC superfamily, is dependent on electrostatic interactions between the negatively charged Asp 57 and Asp 149 residues in extracellular loops 2 and 7, and the positively charged Lys 279 residue in the transmembrane 2-3 linker region of the alpha1-subunit. During gating, Asp 149 and Lys 279 seem to move closer to one another, providing a potential mechanism for the coupling of ligand binding to opening of the ion channel.

Unique general anesthetic binding sites within distinct conformational states of the nicotinic acetylcholine receptor

General anesthesia is a complex behavioral state provoked by the pharmacological action of a broad range of structurally different hydrophobic molecules called general anesthetics (GAs) on receptor members of the genetically linked ligand-gated ion channel (LGIC) superfamily. This superfamily includes nicotinic acetylcholine (AChRs), type A and C gamma-aminobutyric acid (GABAAR and GABACR), glycine (GlyR), and type 3 5-hydroxytryptamine (5-HT3R) receptors. This review focuses on recent advances in the localization of GA binding sites on conformationally and compositionally distinct AChRs. The experimental evidence outlined in this review suggests that: 1. Several neuronal-type AChRs might be targets for the pharmacological action of distinct GAs. 2. The molecular components of a specific GA binding site on a certain receptor subtype are different from the structural determinants of the locus for the same GA on a different receptor subtype. 3. There are unique binding sites for distinct GAs in the same receptor protein. 4. A GA can activate, potentiate, or inhibit an ion channel, indicating the existence of more than one binding site for the same GA. 5. The affinity of a specific GA depends on the conformational state of the receptor. 6. GAs inhibition channels by at least two mechanisms, an open-channel-blocking and/or an allosteric mechanism. 7. Certain GAs may inhibit AChR function by competing for the agonist binding sites or by augmenting the desensitization rate.

A Novel Class of Ligand-gated Ion Channel Is Activated by Zn2+

In mammals, the superfamily of "Cys loop," ligand-gated ion channels (LGICs), is assembled from a pool of more than 40 homologous subunits. These subunits have been classified into four families representing channels that are gated by acetylcholine, serotonin, gamma-aminobutyric acid, or glycine. By searching anonymous genomic sequence data for exons that encode characteristic motifs of the channel subunits, we have identified a novel LGIC that defines a fifth family member. Putative exons were used to isolate a full-length cDNA that encodes a protein of 411 amino acid residues. This protein (ZAC) contains all of the motifs that are characteristic of Cys loop channel subunits but cannot be assigned to any of the four established families on the basis of sequence similarity. Genes for ZAC are present in human and dog but appear to have been lost from mouse and rat genomes. Transcripts of ZAC subunits were detected in human placenta, trachea, spinal cord, stomach, and fetal brain. Transfection of human embryonic kidney cells with ZAC subunit cDNA caused expression of spontaneous current. By screening with a broad range of potential agonists and antagonists, we determined that tubocurarine inhibits the spontaneous current whereas Zn(2+) activates the expressed receptors. The absence of Zn(2+)-activated channels in rats and mice may explain why this fifth member of the LGIC superfamily has evaded detection until now.

Transmembrane segment M2 of glycine receptor as a model system for the pore-forming structure of ion channels.

The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined >pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the >pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer.

Transmembrane segment M2 of glycine receptor as a model system for the pore-forming structure of ion channels.

The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined >pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the >pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer.

Desensitization of neuronal nicotinic receptors.

The loss of functional response upon continuous or repeated exposure to agonist, desensitization, is an intriguing phenomenon if not as yet a well-defined physiological mechanism. However, detailed evaluation of the properties of desensitization, especially for the superfamily of ligand-gated ion channels, reveals how the nervous system could make important use of this process that goes far beyond simply curtailing excessive receptor stimulation and the prevention of excitotoxicity. Here we will review the mechanistic basis of desensitization and discuss how the subunit-dependent properties and regulation of nicotinic acetylcholine receptor (nAChR) desensitization contribute to the functional diversity of these channels. These studies provide the essential framework for understanding how the physiological regulation of desensitization could be a major determinant of synaptic efficacy by controlling, in both the short and long term, the number of functional receptors. This type of mechanism can be extended to explain how the continuous occupation of desensitized receptors during chronic nicotine exposure contributes to drug addiction, and highlights the potential significance of prolonged nAChR desensitization that would also occur as a result of extended acetylcholine lifetime during treatment of Alzheimer's disease with cholinesterase inhibitors. Thus, a clearer picture of the importance of nAChR desensitization in both normal information processing and in various diseased states is beginning to emerge.

Putative histamine-gated chloride channel subunits of the insect visual system and thoracic ganglion.

Histamine-gated chloride channels, members of the ligand-gated ion channel superfamily, are thought to be peculiar for arthropods. Their cognate ligand, histamine, is the transmitter of all arthropod photoreceptors and of thoracic mechanoreceptors. To identify putative histamine-gated chloride channel subunits we scanned the Drosophila genome for putative ligand-gated chloride channel subunits and found 12 candidate genes. We found four groups of transcripts based on their expression pattern. Only members of the last group show an expression pattern that is consistent with our knowledge about histamine-gated chloride channels in insects. In the brain these transcripts (Dm HA-Cl I and II) are exclusively present in interneurones postsynaptic to photoreceptors. Within the lamina (the first visual ganglion) only the L1-L3 neurones are labelled. The lack of non-photoreceptor dependent staining in the brain indicates that mechanosensory transmission differs between the head and the thorax/abdomen, and that the receptors responding to brain-intrinsic histaminergic cells use different signalling pathways. The putative histamine-gated chloride channels show the greatest homology mammalian glycine receptors. These ion-channels are the first specific molecular markers for postsynaptic cells in the insect visual system, thus representing ideal tools to study its physiology and development.

Immunohistochemical localisation of nicotinic acetylcholine receptor subunits in human cerebellum

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel superfamily composed of α and β subunits with specific structural, functional and pharmacological properties. In this study we have used immunohistochemistry to investigate the presence of nicotinic acetylcholine receptor subunits in human cerebellum. Tissue was obtained at autopsy from eight adult individuals (aged 36–56 years). Histological sections were prepared from formalin-fixed paraffin-embedded material. α3, α4, α6, α7, β2, and β4 subunits were present in this brain area associated with both neuronal and non-neuronal cell types. Most Purkinje cells were immunoreactive for all the above subunits, but most strongly for α4 and α7. A proportion of granule cell somata were immunoreactive for all subunits except α3. Punctate immunoreactivity in Purkinje cell and granule cell layers was evident with antibodies against α3, α4, α6, and α7 in parallel with synaptophysin immunoreactivity, suggesting the presence of these subunits on nerve terminals in the human cerebellum. All subunits were present in the dentate nucleus associated with neurones and cell processes. Strong immunoreactivity of neuropil in both the molecular and granule cell layers and within the dentate nucleus was noted with α4, α7 and β4 subunits. Astrocytes and astrocytic cell processes appeared to be immunoreactive for α7 and cell processes observed in white matter, also possibly astrocytic, were immunoreactive for β2. Immunoreactivity to all subunits was noted in association with blood vessels. We suggest that nicotinic acetylcholine receptor subunits may be involved in the modulation of cerebellar activity. Further investigations are warranted to evaluate the participation of nicotinic acetylcholine receptors in cerebellar pathology associated with both developmental and age-related disorders.

NMR Structures of the Second Transmembrane Domain of the Human Glycine Receptor α1 Subunit: Model of Pore Architecture and Channel Gating

Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR α1 subunit are unique in that they form functional homopentameric channels with conductance characteristics nearly identical to those of an authentic receptor (Opella, S. J., J. Gesell, A. R. Valente, F. M. Marassi, M. Oblatt-Montal, W. Sun, A. F. Montiel, and M. Montal. 1997. Chemtracts Biochem. Mol. Biol. 10:153–174). Using NMR and circular dichroism (CD), we determined the high-resolution structures of the TM2 segment of human α1 GlyR and an anesthetic-insensitive mutant (S267Y) in dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. The NMR structures showed right-handed α-helices without kinks. A well-defined hydrophilic path, composed of side chains of G2′, T6′, T10′, Q14′, and S18′, runs along the helical surfaces at an angle ∼10-20° relative to the long axis of the helices. The side-chain arrangement of the NMR-derived structures and the energy minimization of a homopentameric TM2 channel in a fully hydrated DMPC membrane using large-scale computation suggest a model of pore architecture in which simultaneous tilting movements of entire TM2 helices by a mere 10° may be sufficient to account for the channel gating. The model also suggests that additional residues accessible from within the pore include L3′, T7′, T13′, and G17′. A similar pore architecture and gating mechanism may apply to other channels in the same superfamily, including GABAA, nACh, and 5-HT3 receptors.

Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels.

Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of α1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2'Δ) GlyR, had mean inward chord conductances (at −60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance–voltage relationship linear (14 pS at both 60 and −60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

Glycine receptors: lessons on topology and structural effects of the lipid bilayer.

The members of the superfamily of nicotinicoid receptors, sometimes referred to as the ligand-gated ion channel superfamily (LGICS), are essential mediators in the propagation of electrical signals between cells at neuronal and neuromuscular synapses. Given the significant sequence and proposed topological similarities between family members, the structural architecture of any one of these neuroreceptors is believed to be archetypic for the family of ligand-gated channels. We have focused our biophysical studies on the glycine receptor (GlyR) since homomeric expression of just the alpha1 chain of the receptor is sufficient to reconstitute native-like activity when expressed in heterologous cells, and we have successfully overexpressed and purified relatively large quantities of this receptor. Our CD data suggests that the historical four transmembrane helix topology model for nicotinicoid receptors may be erroneous. Proteolytic studies as well as chemical modification studies coupled with mass spectroscopy (MS) have provided additional evidence that this model may be inappropriate. While we suggest a novel topological model for the superfamily of nicotinicoid receptors, the absence of high resolution data for the transmembrane regions of these ion channels precludes further refinement of this model. In addition, we observe structural changes in the recombinant alpha1 GlyR as a function of bilayer composition, suggesting that these receptors may be dynamically modulated by cellular control over the properties of the plasma membrane.