Control of acetylcholine receptor mobility and distribution in cultured muscle membranes. A fluorescence study

The molecular control of the distribution and motion of acetylcholine receptors in the plasma membrane of developing rat myotubes in primary cell culture was investigated by fluorescence techniques. Acetylcholine receptors were marked with tetramethylrhodamine-labeled α-bungarotoxin and lateral molecular motion in the membrane was measured by the fluorescence photobleaching recovery technique. Three types of experiments are discussed: (I) The effect of enzymatic cleavages, drugs, cross-linkers, and physiological alterations on the lateral motion of acetylcholine receptors and on the characteristic distribution of acetylcholine receptors into patch and diffuse areas. (II) Observation of the distribution and/or motion of fluorescence-labeled concanavalin A receptors, lipid probes, cell surface protein, and stained cholinesterase in acetylcholine receptor patch and diffuse areas. (III) The effect of a protein synthesis inhibitor and electrical stimulation on membrane incorporation of new acetylcholine receptors.Some of the main conclusions are: (a) acetylcholine receptor lateral motion is inhibited by concanavalin A plant lectin and by anti-α-bungarotoxin antibody, but marginally enhanced by treatment with a local anesthetic; (b) patches are stabilized by an immobile cellular structure consisting of molecules other than the acetylcholine receptors themselves; (c) this structure is highly selective for acetylcholine receptors and not for other cell membrane components; (d) acetylcholine receptor patch integrity and diffuse area motion are independent of direct metabolic energy requirements and are sensitive to electrical excitation of myotube; (e) lipid molecules can move laterally in both acetylcholine receptor patches and diffuse areas; and (f) acetylcholine receptor lateral motion in diffuse areas and immobility in patch areas are not altered by specific agents which are known to affect extrinsic cell surface proteins, or cytoplasmic microfilaments and microtubules.     

Solubilization of muscarinic acetylcholine receptors of bovine brain

The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [³H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors.     

Inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera from animals with experimental autoimmune myasthenia gravis

Conditions are described for an assay that allows the percent inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera and monovalent antigen-binding fragments of antibody molecules (Fab) to be determined. Anti-Torpedo californica acetylcholine-receptor antisera, prepared in New Zealand White rabbits and Lewis rats, were tested for the ability to inhibit [¹²⁵I]-α-bungarotoxin binding to membrane-associated and detergent-solubilized T californica acetylcholine receptors. Similar inhibition studies were performed using rabbit antisera and antigen-binding fragments prepared against each of the four acetylcholine receptor subunits. Antisera and antigen-binding fragments prepared against intact receptor could inhibit a maximum of 50% of the α-bungarotoxin binding to solubilized receptor. The results using monovalent antigen-binding fragments indicated that the inhibition was not due to antibody-mediated aggregation of receptor molecules. Rabbits and rats immunized with receptor denatured by sodium dodecyl sulfate all produced antisera that could bind to nondenatured receptor, but none of these animals developed experimental autoimmune myasthenia gravis. These results suggest that the antigenic determinants present on acetylcholine receptors responsible for induction of experimental auto-immune myasthenia gravis are lost with sodium dodecyl sulfate denaturation. A strong correlation was also observed between the presence of experimental autoimmune myasthenia gravis in rats and rabbits and the ability of the antisera from these animals to inhibit 50% of α-bungarotoxin binding to solubilized acetylcholine receptors.     

Solubilization and characterization of CCK receptors from mouse pancreas

To study the characteristics of the CCK receptor, plasma membranes were prepared from mouse pancreatic acini, and CCK receptors solubilized with 1% digitonin. To measure hormone binding, the solubilized receptors were incubated with 125I-CCK at 4 degrees C and the hormone-receptor complex was precipitated with 10% polyethylene glycol. Specific 125I-CCK binding by the solubilized CCK receptor was compared to that by the plasma membrane-bound CCK receptor. Both the solubilized and the membrane-bound receptor displayed optimal binding at an acidic pH (between 6.0 and 7.0) and showed a similar sensitivity to monovalent and divalent cations. The solubilized receptors preserved their relative specificity for CCK molecules: CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. However, the soluble CCK receptor had a lower binding affinity than plasma membrane-bound receptor. Solubilized receptors preserved their relative specificity for inhibitors of CCK binding and action: dibutyryl cyclic GMP greater than N-CBZ-tryptophan greater than proglumide. Solubilized receptors had affinities for these antagonists that were similar to receptors on intact plasma membranes. These data indicate, therefore, that the specific binding properties of the CCK receptor are inherent to the solubilized glycoprotein molecules.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.     

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.     

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002     

Crystallization of kappa-bungarotoxin: preliminary X-ray data obtained from the venom-derived protein.

kappa-Bungarotoxin is a 66 residue polypeptide found in the venom of the Taiwanese banded krait, Bungarus multicinctus. It binds tightly to neuronal nicotinic acetylcholine receptors and inhibits nerve transmission mediated by these postsynaptic receptors. It is related, by similarity in amino acid sequence, to alpha-bungarotoxin and other alpha-neurotoxins, but differs sharply in physiologic action. The alpha-neurotoxins inhibit nerve transmission in nicotinic acetylcholine receptors associated with vertebrate skeletal muscle and fish electric organs. The kappa-neurotoxins inhibit nerve transmission in neuronal nicotinic acetylcholine receptors such as those found in chick ciliary ganglia. The kappa-neurotoxins display a low level of interaction with receptors that are strongly affected by alpha-neurotoxins, but alpha-neurotoxins are completely without effect on receptors that are affected by kappa-bungarotoxin. The structural basis for this physiologic differentiation is not known. Crystals of kappa-bungarotoxin have now been obtained that diffract to at least 2.3 A. These crystals are hexagonal, space group P6, and have dimensions of a = b = 80.2 A, c = 39.6 A, and angles of alpha = beta = 90 degrees and gamma = 120 degrees. Each unit cell contains 12 molecules of the 66 residue protein or two molecules per asymmetric unit. Comparison of the structure of kappa-bungarotoxin, which will result from further diffraction analysis of these crystals, with the structures of the alpha-neurotoxins that have been determined may provide information on the structural basis of physiologic action in these acetylcholine receptor antagonists.     

Intrinsic lattice formation by the ryanodine receptor calcium-release channel

Recent theoretical analysis of a model lattice of interacting transmembrane receptor proteins has indicated that such clustering in the membrane could provide a novel mechanism for regulating receptor signalling in cells. It has been calculated that cooperative interactions between receptors organized into a cluster, or array, in the membrane would dramatically increase their sensitivity to activation by ligand. Sensitivity to ligand would increase with the extent of spread of activity within the receptor lattice. Hence, formation of extensive receptor lattices in the membrane would allow a large population of receptors to be simultaneously switched on, or off, by a very small change in ligand concentration. We show here that lattice formation is an intrinsic property of an integral membrane protein, the ryanodine-sensitive calcium-release channel (RyR) of endoplasmic reticulum. The purified protein spontaneously assembled into two-dimensional lattices in solution, enabling the construction of a 25 A projection map that identifies the mode of interaction between RyR oligomers. Our observations on the RyR provide a new perspective on various properties of cell signalling via this and other receptors.     

Dissociation between adenosine receptors and adenylate cyclase in the smooth muscle of guinea pig myometrium

We have previously demonstrated that adenosine causes contraction of guinea-pig myometrium in a fashion consistent with the presence of a purinergic receptor of the A1 subtype. Incubation of guinea-pig uterine smooth muscle membranes with the stable adenosine analogue [3H]cyclohexyladenosine [( 3H]CHA) resulted in rapid, reversible association of radioligand to saturable sites. The affinity (KD) of the receptor for [3H]CHA determined from kinetic experiments (3.14 nM) is in good agreement with that determined in saturation experiments (KD = 4.5 nM). Scatchard analysis of specific [3H]CHA binding (Bmax = 79 fmol/mg protein) is consistent with a single class of binding sites for [3H]CHA. Computer analysis of competition of [3H]CHA binding by the stereoisomers of phenylisopropyl adenosine, R-PIA (KI = 5.3 nM) and S-PIA (KI = 69 nM), as well as the 5'-substituted analogue, ethylcarboxamide adenosine (NECA; KI = 4.2 nM) suggest that [3H]CHA binding occurs to a single class of receptors of the AI subtype. Contractile studies employing these agents reveal that the relative order of potency, based on ED50 values, correlates well with the relative order of competition of agonist binding, based on equilibrium binding constants. Direct assay of myometrial adenylate cyclase failed to show that adenosine receptors in this smooth muscle are coupled to adenylate cyclase. We conclude here that a smooth muscle adenosine receptor is not coupled to adenylate cyclase, yet subserves muscle contraction. These data are important in light of recent attempts to classify adenosine receptors as dual regulators of adenylate cyclase.     

The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton.     

Minimal RNA Aptamer Sequences That Can Inhibit or Alleviate Noncompetitive Inhibition of the Muscle-Type Nicotinic Acetylcholine Receptor

Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10¹¹ neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.     

Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.     

Functional stoichiometry at the nicotinic receptor. The photon cross section for phase 1 corresponds to two bis-Q molecules per channel

These experiments examine changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed. Voltage- clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-Bis-Q. A 1-microsecond laser flash photoisomerizes some trans-Bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans leads to cis photoisomerization. Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of Bis-Q solutions caused by flashes. At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. As a partial control for changes in the photochemistry caused by binding of Bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule.     

Studies of reversible and irreversible interactions of an alkylating agonist with Torpedo californica acetylcholine receptor in membrane-bound and purified states.

The interaction of a cholinergic depolarizing agent, bromoacetylcholine, with acetylcholine receptor (AcChR) enriched membrane fragments and Triton-solubilized, purified AcChR from Torpedo californica has been studied. The reagent bound to membrane-bound AcChR reversibly with an apparent dissociation constant of 16 +/- 1 nM at equilibrium. This 600-fold higher affinity for the receptor than found from physiological studies [Kact congruent to 10 micrometers; Karlin, A. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 1847--1853] can be attributed to a ligand-induced affinity change of the membrane-bound receptor upon preincubation with bromoacetylcholine. At equilibrium [3H]bromoacetylcholine, like acetylcholine, bound to half the number of alpha-bungarotoxin sites present in the preparation without apparent positive cooperativity, and this binding was competitively inhibited by acetylcholine. In the presence of dithiothreitol, [3H]bromoacetylcholine irreversibly alkylated both membrane-bound and solubilized, purified acetylcholine receptor, with a stoichiometry identical with that for reversible binding. NaDodSO4-polyacrylamide gel electrophoresis of the labeled acetylcholine receptor showed that only the 40 000-dalton subunit contained the label. From these results it is concluded that the 40 000-dalton subunit represents a major component of the agonist binding site of the receptor.     

Adenosine A₂A and A₂B receptors are both required for adenosine A₁ receptor-mediated cardioprotection

All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A(2A) and/or A(2B) receptors modulate adenosine A(1) receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A(2A) knockout (KO), and A(2B)KO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A(2A) receptor agonist CGS-21680 (200 nM) and the A(2B) agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A(2A) or A(2B) receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A(2A) and A(2B) receptors are required for adenosine A(1) receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.     

Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation [corrected and republished article originally printed in J Cell Biol 1988 May;106(5):1723-34]

Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation.     

Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors)

New techniques for investigating chemical reactions on cell surfaces in the microsecond-to-millisecond time region are described. Reactions mediated by membrane-bound neurotransmitter receptors that control signal transmission between 10¹² cells of the nervous system are taken as an example. Cells with receptors on their plasma membrane are equilibrated with photolabile, biologically inactive precursors of the neurotransmitters. Photolysis of these compounds releases free neurotransmitter that interacts with the receptors, leading to the transient opening of transmembrane receptor-formed channels that are permeant to small inorganic ions. The current thus induced can be measured. The technique can be used to measure the elementary steps of the receptor-mediated reactions. To illustrate the approach it was shown that an understanding of the mechanism of inhibition of the nicotinic acetylcholine receptor by the drug cocaine was obtained and led to the first proof that compounds exist that alleviate the inhibition.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.