Cross-Linking-Mass Spectrometry Studies of Cholesterol Interactions with Human α1 Glycine Receptor.

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Cholesterol at compositions below ∼33 mol percent has been shown to have negligible chemical activity, suggesting that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol/lipid ratios above and below the cholesterol activity threshold with equivalent aliquots of azi-cholesterol, a photoactivatable nonspecific cross-linker. After photoactivation, cross-linked cholesterol-GlyR was trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS), and sites of direct covalent attachment to peptides were refined by targeted MS/MS. Differential patterns of dozens of cholesterol-GlyR cross-links were identified in these comparative studies, with sites of cross-linking found primarily in the fourth transmembrane helix and extramembranous connecting loops and mapping the lipid-accessible surface of the receptor. Unique cross-linking observed in both reduced and elevated cholesterol composition suggests different apo-state structural conformations of GlyR as a function of cholesterol concentration and, in the latter studies, identified potential specific binding sites for cholesterol in the receptor.

Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster.

Chloroquine, an amino quinolone derivative commonly used as an anti-malarial drug, is known to impart an unpleasant taste. Little research has been done to study chloroquine taste in insects, therefore, we examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. We identified the antifeedant effect of chloroquine by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. We discovered that two molecular sensors, GR22e and GR33a, act as chloroquine receptors, and found that chloroquine-mediated activation of GRNs occurs through S-type sensilla. At the same time, we successfully recapitulated the chloroquine receptor by expressing GR22e in ectopic gustatory receptor neurons. We also found that GR22e forms a part of the strychnine receptor. We suggest that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials.

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor.

Herein, we identify the coordination environment of Cu²(+) in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²(+) and Cu²(+) significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²(+) can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²(+) is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²(+) coordination and stoichiometry. We show that Cu²(+) has one binding site per α1 subunit, and that five Cu²(+) can be coordinated per GlyR. Cu²(+) binds to E192 and H215 in each subunit of GlyR with a 40 µM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²(+) coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²(+) site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.

RNA editing produces glycine receptor alpha3(P185L), resulting in high agonist potency.

The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

Isolation and characterization of an alpha 2-type zebrafish glycine receptor subunit.

The complementary DNA for a novel alpha subunit of the glycine receptor, alphaZ2, was isolated from a zebrafish adult brain library. The molecular characteristics, phylogenetic relationships and messenger RNA length of this alphaZ2 subunit show it to be an alpha2-type glycine receptor subunit isoform. The leader peptide however, diverges from those of known glycine receptor alpha isoforms. Recombinantly expressed in Xenopus oocytes, alphaZ2 formed functional glycine receptor channels. These homomeric channels were activated by glycine and taurine, with apparent affinities similar to those reported for zebrafish alphaZ1 glycine receptor, and were also effectively antagonized by nanomolar concentrations of strychnine. However, during prolonged applications of agonists, ionic currents of alphaZ2 receptor channels declined to a much lower steady-state level than those of alphaZ1, indicating different desensitization properties. Analysis of messenger RNA revealed that alphaZ2 is specifically expressed in adult brain tissue and present in both adult and embryonic zebrafish. This report contributes to the characterization of the diversity of glycine receptor isoforms in vertebrates.

High-performance liquid chromatographic purification of extremely hydrophobic peptides: transmembrane segments.

Transmembrane peptides of integral membrane proteins often exhibit extremely high hydrophobicity. Therefore, the solubility of such peptides in solvents commonly used in HPLC is usually very low and the interaction with generally applied stationary phases such as silica gel or C18 reversed phases appears to be extremely strong, which makes the characterization and purification of these peptides difficult. The analytical characterization and preparative separation of the synthesized M1 transmembrane sequence of the inhibitory glycine receptor M(r) 48,000 subunit and some of its fragments is shown. M1 and its larger fragments could be dissolved in a dichloromethane-hexafluoro-2-propanol mixture containing a trace amount of pyridine for their separation on a C4 phase by employing linear two-component gradients of formic acid-2-propanol and formic acid-water with ratios up to 4:1 (v/v). Conditions to avoid formylation of the peptides are indicated.

Baculovirus-driven expression and purification of glycine receptor alpha 1 homo-oligomers.

The glycine receptor is a ligand-gated anion channel protein of postsynaptic membranes. We expressed a homo-oligomeric receptor composed of human alpha 1 subunits in Spodoptera frugiperda cells by infection with a recombinant Autographa californica nuclear polyhedrosis virus. A substantial fraction of the recombinant receptor was incorporated as a functional channel protein into the cell's plasma membrane at expression levels 4- to 30-fold higher than in other eukaryotic heterologous expression systems or native rat spinal cord membranes, respectively. Upon detergent solubilization, the alpha 1 receptor was found to exist in a predominantly monodisperse state and could be affinity-purified to near homogeneity. This preparation is a potential starting point for future crystallisation studies.

Modulation of inhibitory glycine receptors by phosphorylation by protein kinase C and cAMP-dependent protein kinase.

Recent evidence has suggested a role for phosphorylation in the regulation of ligand-gated ion channels. We have recently shown (Ruiz-Gómez, A., Vaello, M., Valdivieso, F., and Mayor, F., Jr. (1991) J. Biol. Chem. 266, 559-566) that the inhibitory glycine receptor (GlyR) alpha subunit is phosphorylated in vitro by protein kinase C (PKC). In this report we further show that alpha subunits of the GlyR can also be phosphorylated by cAMP-dependent protein kinase (PKA) in an in vitro assay. Moreover, incubation of intact rat spinal cord neurons with specific PKC or PKA activators leads to increased phosphorylation of the GlyR alpha subunits, strongly suggesting a physiological role in its functional modulation. The role of protein phosphorylation in modulating GlyR channels was explored in Xenopus oocytes injected with poly (A)+ mRNA isolated from nervous tissue. The treatment of oocytes with phorbol esters or dibutyryl cAMP resulted in a decrease or an enhancement, respectively, of glycine-evoked currents. Our results show that the GlyR can be phosphorylated in vivo in response to activation of either PKC or PKA with opposite functional consequences, suggesting that neurotransmitters affecting the activity of such kinases could profoundly alter glycine-mediated neuronal signaling and modulate synaptic efficacy.

Functional expression and purification of a homomeric human alpha 1 glycine receptor in baculovirus-infected insect cells.

The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.