Sequence analysis of the 5'-untranslated region of the human H1 histamine receptor-encoding gene.

A clone containing the H1 histamine receptor (H1HR)-encoding gene was isolated from a human genomic DNA library. The 5'-UTR of the H1HR gene reported here differs upstream from bp -142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894-901]. PCR amplification utilizing primer pairs derived from the 5'-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5'-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5'-UTR of H1HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.

Stable allosteric binding of m1-toxin to m1 muscarinic receptors.

m1-Toxin was found to slow the dissociation of [3H]N-methyl-scopolamine (NMS) and [3H]pirenzepine from m1 muscarinic receptors expressed in the membranes of Chinese hamster ovary cells. When toxin-NMS-receptor complexes were formed in membranes and then dissolved in digitonin, or when these complexes were formed in solution, the toxin completely stopped the dissociation of [3H]NMS for 6 hr at 25 degrees C. Toxin-receptor complexes formed in membranes or in solution were also highly stable in solution at 25 degrees, as shown by the ability of the toxin to prevent the binding of [3H]quinuclidinyl benzilate (QNB). [3H] QNB-receptor complexes were equally stable, whereas unliganded soluble receptors lost most of their ability to bind QNB within an hour. Toxin-receptor complexes could be partially dissociated by incubation at 37 degrees in the presence of digitonin and [3H]QNB, and the freed receptors were then labeled. These results demonstrate that m1-toxin binds allosterically and pseudoirreversibly to m1 receptors, and that the toxin can stabilize the outward-facing pocket of m1 receptors which contains and binds competitive antagonists. The allosteric nature of the binding of m1-toxin should prove to be useful for such unusual purposes as stabilizing the binding of readily reversible and/or nonselective ligands specifically to m1 receptors, for purifying labeled or unlabeled receptors by affinity techniques which recognize the toxin, for recognizing receptors with genetically or biochemically altered primary binding sites, and for stabilization of the native conformation of m1 receptors for structural studies.

Use of m1-toxin as a selective antagonist of m1 muscarinic receptors.

m1-Toxin is the only ligand which is known to bind specifically to the extracellular face of genetically defined m1 muscarinic receptors; it binds pseudoirreversibly. A variety of studies were performed to evaluate the usefulness of m1-toxin as a selective antagonist of m1 receptors. Exposure of slices of the rat cerebral cortex to m1-toxin in physiological buffer blocked the subsequent binding of 1.0 nM [3H]pirenzepine to m1 receptors in the slices. The toxin also blocked 70% of carbachol-stimulated turnover of radiolabeled inositol phosphates in hippocampal slices. Autoradiographs showed that m1-toxin bound to sections of once-frozen tissue and blocked the binding of [3H]quinuclidinyl benzilate to regions of the rat brain rich in m1 receptors. The toxin blocked the binding of [3H]antagonists to pure m1 receptors on the surface of living Chinese hamster ovary cells, but did not block intracellular receptors. In membrane preparations from the rat cortex and hippocampus the toxin blocked the binding of [3H] antagonists to m1 receptors quantitatively and selectively, but had no effect on binding sites for [3H]nicotine. Subsaturating amounts of the toxin bound to m1 receptors in membranes at 4 degrees C in less than 30 sec. Low concentrations of m1-toxin blocked m1 receptors in solution in digitonin but had no effect on separate preparations of pure m2, m3, m4 or m5 receptors. Thus m1-toxin appears to be a very useful antagonist for m1 receptors in intact tissue, on isolated cells, in membranes and in solution, in a variety of media.

Purification and properties of m1-toxin, a specific antagonist of m1 muscarinic receptors.

The venom of the Eastern green mamba from Africa, Dendroaspis angusticeps, was found to block the binding of 3H-quinuclidinyl benzilate to pure m1 and m4 muscarinic ACh receptors expressed in Chinese hamster ovary cells. The principal toxin in the venom with anti-m1 muscarinic activity was purified by gel filtration and reversed-phase HPLC. This toxin has 64 amino acids, a molecular mass of 7361 Da, and an isoelectric point of 7.04. Its cysteine residues are homologous with those in curare-mimetic alpha-neurotoxins, and with those in fasciculin, which inhibits AChE. At low concentrations the toxin blocked m1 receptors fully and pseudoirreversibly while having no antagonist activity on m2-m5 receptors; the toxin is therefore named "m1-toxin." At higher concentrations m1-toxin interacted reversibly with m4 receptors, and half of the toxin dissociated in 20 min at 25 degrees C. The affinity of m1-toxin is therefore much higher for m1 than for m4 receptors. By comparison with m1-toxin, pirenzepine has sixfold higher affinity for m1 than for m4 receptors. Autoradiographs of muscarinic receptors in the rat brain demonstrated that m1-toxin blocked the binding of 2 nM 3H-pirenzepine only in regions known to bind m1-specific antibodies. Thus, m1-toxin is a much more selective ligand than pirenzepine for functional and binding studies of m1 muscarinic receptors.

m1-toxin.

The venom of the Eastern green mamba from Africa, Dendroaspis angusticeps, contains a number of toxins which block the binding of 3H-antagonists to genetically-defined m1 and m4 muscarinic acetylcholine receptors. Most of the anti-muscarinic activity of the venom is due to the presence of a newly-isolated toxin, "m1-toxin", which has 64 amino acids and a molecular mass of 7361 Daltons. At present m1-toxin is the only ligand which is known to be capable of fully blocking m1 receptors without affecting m2-m5 receptors. It binds very rapidly, specifically and pseudoirreversibly to the extracellular face of m1 receptors on cells, in membranes or in solution, whether or not the primary receptor site is occupied by an antagonist. Bound toxin can either prevent the binding and action of agonists or antagonists, or prevent the dissociation of antagonists. The toxin is useful for identifying m1 receptors during anatomical and functional studies, for recognizing and stabilizing receptor complexes, and for occluding m1 receptors so that other receptors are more readily studied.