Mechanisms for picrotoxin block of alpha2 homomeric glycine receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.     

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.     

Mechanisms for picrotoxinin and picrotin blocks of alpha2 homomeric glycine receptors

Contrary to its effect on the gamma-aminobutyric acid type A and C receptors, picrotoxin antagonism of the alpha1 homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and nonselective between the picrotoxin components picrotoxinin and picrotin. Picrotoxin antagonism of the embryonic alpha2 homomeric GlyR is known to be use-dependent and reflects a channel-blocking mechanism, but the selectivity of picrotoxin antagonism of the embryonic alpha2 homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin- and picrotoxinin-induced inhibition of currents, which were evoked by the activation of alpha2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents, picrotin had a 30 times higher IC50 than picrotoxinin. Picrotin-evoked inhibition displayed voltage dependence, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentration-dependent manner, indicating that these picrotoxin components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were co-applied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but both could be trapped at their binding site when the channel closed during glycine dissociation. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking alpha2 homomeric GlyR.     

Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective alpha1 homomeric glycine receptor (alpha1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1'E GlyR) was sufficient to convert the channels to select cations over anions with P(Cl)/P(Na) = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2'Delta mutant GlyR retained its anion selectivity (P(Cl)/P(Na) = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (P(Cl)/P(Na) = 27.9). When the A-1'E and the P-2'Delta mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (P(Cl)/P(Na) = 0.13). The SDM GlyR was also Ca(2+) permeable (P(Ca)/P(Na) = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca(2+)-permeable channel (P(Ca)/P(Na) = 0.73), without drastically altering monovalent charge selectivity (P(Cl)/P(Na) = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca(2+) permeability (P(Ca)/P(Na) = 0.92), with little effect on monovalent selectivity (P(Cl)/P(Na) = 0.19). Estimates of the minimum pore diameter of the A-1'E, SDM, SDM+R19'A, and SDM+R19'E GlyRs revealed that these pores are larger than the alpha1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.     

Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships

GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundstr?m, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to alpha(1) GlyR and rho(1) GABAR, the rounded end of PTX accepts several H-bonds from Thr(6')s, while the elongated end enters ring R(2'). The lack of H-bond donors on the side chains of Phe(6')s (beta GlyR) and Met(6')s (rho(2) GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro(2')s/Ala(2')s in GABARs, but fit a more hydrophilic ring with Gly(2')s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations.     

Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel

The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.     

Probing the topology of the glycine receptor by chemical modification coupled to mass spectrometry

Tetranitromethane (TNM), a small aqueous reagent that specifically modifies solvent-accessible tyrosine residues to o-nitrotyrosine, was used to probe the topology of the GlyR. Homomers of human alpha1 GlyR were recombinantly expressed via a baculovirus system, affinity-purified, and reconstituted in lipid vesicles of defined composition. The native-like reconstituted receptors were then reacted with TNM, and GlyR reaction products were isolated by SDS-PAGE. After proteolytic digestion, TNM-labeled residues were identified using mass spectrometry by observing the mass shift corresponding to the nitrate moiety. In this manner, we have identified TNM modifications of tyrosine residues at positions 24, 75, 78, 161, 223, and 228 in the receptor. Of significance, nitrations at Tyr 223 and Tyr 228 occur within the first putative transmembrane helix (M1) of the receptor, and their labeling suggests a non-helical secondary structure for M1 for the glycine receptor. In a previously published report [Leite et al. (2000) J. Biol. Chem. 275, 13683], we also identified proteolytic cleavage sites within M1. Taken together, these studies support a topological model where the "historical" M1 segment cannot be entirely alpha-helical and may contain an extramembranous surface loop. Furthermore, we have also identified a tyrosine modification (Tyr 161) within a region of the N-terminal domain critical in agonist and antagonist binding.     

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABA(A)R) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.     

Mutations within the agonist-binding site convert the homomeric α1 glycine receptor into a Zn2+-activated chloride channel

The divalent cation Zn²⁺ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (10 µM) have an inhibitory effect. Mutational studies have localized the Zn²⁺ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR α-subunits. Here, we examined the ZZn²⁺ sensitivity of different mutations within the agonist binding site of the homomeric α1-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that 6 substitutions within the ligand-binding pocket result in a total loss of Zn²⁺ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (α1R65A, α1R131A), or of the aromatic residue phenylalanine 207 by histidine (α1F207H), converted the α1 GlyR into a chloride channel that was activated by Zn²⁺ alone. Dose-response analysis of the α1F207H GlyR disclosed an EC50 value of 1.2 µM for Zn²⁺ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the α1 subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn²⁺-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.     

GABA-stimulated 36Cl- influx into reconstituted vesicles with purified GABAA/benzodiazepine receptor complex

Solubilized and Purified gamma-aminobutyric acid (GABA)A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl- influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl- influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABAA/benzodiazepine receptor complex and Cl- channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl- channel also exhibited GABA-stimulated 36Cl- influx. This GABA-stimulated influx of 36Cl- was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABAA receptor antagonist. These results strongly suggest that GABAA receptor is directly coupled with Cl- channel, whereas benzodiazepine receptor may be functionally coupled with GABAA receptor and modulates the GABA-stimulated Cl- influx through GABAA receptor. The present results also indicate that the purified GABAA receptor complex is coupled with Cl- channel and possesses functional characteristics as GABAA receptor.     

Microsecond Simulations Indicate that Ethanol Binds between Subunits and Could Stabilize an Open-State Model of a Glycine Receptor

Cys-loop receptors constitute a superfamily of ion channels gated by ligands such as acetylcholine, serotonin, glycine, and γ-aminobutyric acid. All of these receptors are thought to share structural characteristics, but due to high sequence variation and limited structure availability, our knowledge about allosteric binding sites is still limited. These sites are frequent targets of anesthetic and alcohol molecules, and are of high pharmacological importance. We used molecular simulations to study ethanol binding and equilibrium exchange for the homomeric α1 glycine receptor (GlyRα1), modeled on the structure of the Gloeobacter violaceus pentameric ligand-gated channel. Ethanol has a well-known potentiating effect and can be used in high concentrations. By performing two microsecond-scale simulations of GlyR with/without ethanol, we were able to observe spontaneous binding in cavities and equilibrium ligand exchange. Of interest, it appears that there are ethanol-binding sites both between and within the GlyR transmembrane subunits, with the intersubunit site having the highest occupancy and slowest exchange (∼200 ns). This model site involves several residues that were previously identified via mutations as being crucial for potentiation. Finally, ethanol appears to stabilize the GlyR model built on a presumably open form of the ligand-gated channel. This stabilization could help explain the effects of allosteric ligand binding in Cys-loop receptors.     

Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.     

Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-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 V13'T in M2 together with neutralization of glutamate residues (E-1'A) 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 alpha7 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.     

Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily

The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-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 V13'T in M2 together with neutralization of glutamate residues (E-1'A) 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 alpha7 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.     

Homology modeling and molecular dynamics simulations of the alpha1 glycine receptor reveals different states of the channel

Homology modeling is used to build initial models of the transmembrane domain of the human alpha1 glycine receptor (GlyR) based on the most recently published refined structure of nAChR (PDB ID: 2BG9). Six preliminary GlyR models are constructed using two different approaches. In one approach, five different homopentamers are built by symmetric assembly of alpha1 GlyR subunits using only one of the five unique chains of nAChR as a template. In a second approach, each nAChR subunit serves as a template for an alpha1 GlyR subunit. All six initial GlyR constructs are then embedded into a hydrated POPC lipid bilayer and subjected to molecular dynamics simulation for at least six nanoseconds. Each model is stable throughout the simulation, and the final models fall into three distinct categories. Homopentameric GlyR bundles using a single alpha nAChR subunit as a template appear to be in an open conformation. Under an applied external potential, permeation of Cl(-) ions is observed within several ns in a channel built on an alpha chain. Model channels built on non-alpha chains have a constriction either near the intracellular mouth or more centrally located in the pore domain, both of which may be narrow enough to close the channel and whose locations correspond to putative gates observed in nicotinicoid receptors. The differences between these three general models suggest that channel closure may be effected by either rotation or tangential tilting of TM2.     

The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system

The postsynaptic glycine receptor (GlyR) is a major inhibitory chloride channel protein in the central nervous system. The affinity-purified receptor contains polypeptides of 48 kDa, 58 kDa, and 93 kDa. The 48-kDa (alpha) and 58 kDa (beta) subunits span the postsynaptic membrane in a pentameric arrangement to form the anion channel of the receptor. The 93-kDa polypeptide is cytoplasmically localized and may have an anchoring function. Molecular cloning revealed that different structural characteristics are shared by the membrane-spanning subunits of the GlyR and those of other ligand-gated ion channel proteins. Developmental regulation of the GlyR is characterized by alterations in antagonist binding, heterogeneity of alpha subunits, and increased levels of the 93-kDa polypeptide. Glycine receptor function can be reconstituted by expression of cloned alpha subunits in heterologous cell systems. Positive charges found at the presumed mouths of the GlyR channel appear to be important determinants of ion selectivity. These data establish the anion-conducting GlyR as a homolog of other ligand-gated ion channel proteins and suggest that the diversity of these channels originates from divergent evolution of a primordial channel protein early in phylogeny.     

Distinct properties of glycine receptor β+/α- interface: unambiguously characterizing heteromeric interface reconstituted in homomeric protein

The glycine receptor (GlyR) exists either in homomeric α or heteromeric αβ forms. Its agonists bind at extracellular subunit interfaces. Unlike subunit interfaces from the homomeric α GlyR, subunit interfaces from the heteromeric αβ GlyR have not been characterized unambiguously because of the existence of multiple types of interface within single receptors. Here, we report that, by reconstituting β+/α- interfaces in a homomeric GlyR (αChb+a- GlyR), we were able to functionally characterize the αβ GlyR β+/α- interfaces. We found that the β+/α- interface had a higher agonist sensitivity than that of the α+/α- interface. This high sensitivity was contributed primarily by loop A. We also found that the β+/α- interface differentially modulates the agonist properties of glycine and taurine. Using voltage clamp fluorometry, we found that the conformational changes induced by glycine binding to the β+/α- interface were different from those induced by glycine binding to the α+/α- interface in the α GlyR. Moreover, the distinct conformational changes found at the β+/α- interface in the αChb+a- GlyR were also found in the heteromeric αβ GlyR, which suggests that the αChb+a- GlyR reconstitutes structural components and recapitulates functional properties, of the β+/α- interface in the heteromeric αβ GlyR. Our investigation not only provides structural and functional information about the GlyR β+/α- interface, which could direct GlyR β+/α- interface-specific drug design, but also provides a general methodology for unambiguously characterizing properties of specific protein interfaces from heteromeric proteins.     

Probing the pharmacological properties of distinct subunit interfaces within heteromeric glycine receptors reveals a functional ββ agonist-binding site

Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α(1) subunit by the corresponding residues of the β subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α(1) GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ββ-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.     

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.     

Effect of colchicine on internalization of prolactin in female rat liver: an in vivo radioautographic study

Binding and internalization of 125I-ovine prolactin into hepatocytes of female rats was visualized by the in vivo radioautographic method (Bergeron, J. J. M., G. Levine, R. Sikstrom, D. O'Shaughnessey, B. Kopriwa, N. J. Nadler, and B. I. Posner, 1977, Proc. Natl. Acad. Sci. USA, 745:051-5055). Receptor-mediated internalization of label was observed into lipoprotein-filled vesicles in the Golgi/bile canalicular region of the hepatocyte. Colchicine treatment had no effect on the internalization of label into the lipoprotein-filled vesicles. However, the location of the radio-labeled lipoprotein-filled vesicles was altered from the Golgi/bile canalicular region to subsinusoidal. Radioactive content of hepatocytes decreased as a function of time after injection of 125I-prolactin; however, colchicine treatment markedly retarded this loss of label. Subcellular fractionation experiments indicated that colchicine treatment led to decreased levels of 125I-prolactin accumulation in microsomes but augmented the accumulation of label in the L fraction. It is concluded that in normal female rats prolactin is internalized into lipoprotein-filled vesicles in the Golgi region before degradation of the hormone. Colchicine treatment accumulates labeled lipoprotein-containing vesicles in a subsinusoidal region and retards hormone catabolism. The labeled vesicles observed after colchicine treatment may correspond to the unique vesicles previously observed in the L fraction and found to be enriched in prolactin receptors (Khan, M. N., B. I. Posner, A. K. Verma, R. J. Khan, and J. J. M. Bergeron, 1981, Proc. Natl. Acad. Sci. USA, 78:4980-4981).