Symmetry and dimensions of membrane-bound nicotinic acetylcholine receptors from Torpedo californica electric tissue: rapid rearrangement to two-dimensional ordered lattices

Computer-aided image-averaging methods are applied to different preparations of membrane-bound nicotinic acetylcholine receptor. Circular harmonic averaging (CHA), a novel, reference-independent averaging method developed by W. Kunath and H. Sack-Kongehl [1989) Ultramicroscopy 27:171-184) allows analyzing images of single molecules of the receptor in its native membrane-bound state. The five subunits of the receptor are clearly resolved. At the resolution obtained (approximately 20 A) no differences were observed with resting and agonist-desensitized receptors. A method is proposed for rapidly arranging the acetylcholine receptors to ordered lattices. Depending on the conditions, tetragonal or hexagonal, two-dimensional lattices can be obtained within 2 to 6 days at 4 degrees C. Analysis by CHA shows that the receptor molecules preserve their gross structure and dimensions in these membranes, but that they are randomly oriented. Both lattices, therefore, do not represent true two-dimensional crystals.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.     

Rapid laser flash photoaffinity labeling of binding sites for a noncompetitive inhibitor of the acetylcholine receptor

Photoaffinity labeling of the nicotinic acetylcholine receptor from Torpedo marmorata electric tissue was performed in the presence of cholinergic effectors in the millisecond to second time range by a combination of a stopped-flow apparatus and a high-energy pulse laser. The label applied was [3H]triphenylmethylphosphonium, a lipophilic cation previously shown to be a specific blocker of the acetylcholine receptor ion channel. With the receptor in the resting state most of the label was incorporated into the alpha polypeptide chains. In the presence of agonists and antagonists increasing incorporation into the delta- and (less pronounced) the beta-chain was observed. The time course of this increase had a half-life of about 0.4 s, being slower than receptor activation and channel opening. in the resting, active, and even rapidly desensitized state, the alpha polypeptide chains appear to be the primary targets of the photoaffinity reaction. The action spectrum of the photolabeling has a sharp maximum at lambda = 270 nm and a small-side maximum at lambda = 290 nm. It does not resemble the absorption spectrum of the label and may hint at amino acid side chains as the moieties activated by UV light causing the photolabeling. The effector specificity of the observed slow increase of label incorporation into the delta polypeptide chain was investigated. It does not prove that slow desensitization is the underlying event. The agonists acetylcholine and carbamoylcholine as well as treatment of receptor-rich membranes with phospholipase A2 (but not phospholipase D) triggered labeling of delta, but antagonists such as D-tubocurarine and most conspicuously flaxedil had a similar effect.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [¹²⁵I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor's, , , or subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.Abbreviations AChR Acetylcholine receptor - NDPA [2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]]phenoxy]acetyl - NT-II neurotoxin II     

Radioactive labelling of toxin I from Anemonia sulcata and binding to crayfish nerve in vitro

1. Radioactive derivatives of neurotoxin I (ATX I) from Anemonia sulcata have been synthesized: Iodination of ATX I with 125I yielded a mixture of reaction products from which monoiodo and diiodo ATX I were isolated. 2. 125I-ATX I was shown to bind to the axonal membrane from Astacus leptodactylus main walking nerve. Specificity of binding was shown by saturability of the binding sites and by competitive binding of native and radioactive toxin. 3. Astacus nerve bound 44 fmol of 125I-ATX I/mg nerve (wet weight). The axonal membrane surface of the nerve was determined to be 7800 cm2/g nerve. This amounts to a binding site density of around 35/mu2 axonal surface. Binding was not inhibited by tetrodotoxin, the blocker of the selectivity filter of voltage-dependent sodium channels. 125I-ATX I therefore may bind to the sodium channel-inactivating gate. 4. The affinity of the nerve membrane receptors for 125I-ATX I appears to be voltage-dependent: KD = 5 nM was found with whole crayfish nerves in the presence of tetrodotoxin, KD = 40nM in the absence of tetrodotoxin and an even lower affinity was obtained with axonal membrane fragments isolated from the nerve. Drugs destabilizing the membrane potential, e.g. veratridine, ouabain and sodium azide lowered the affinity or abolished binding completely.     

Location of the polyamine binding site in the vestibule of the nicotinic acetylcholine receptor ion channel

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.     

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.