Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor-1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor-1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor-1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.
The synthesis, biological activity, and molecular modeling of a novel series of substituted 1-(3-pyridylcarbamoyl)indolines are reported. These compounds are isosteres of the previously published indole urea 1 (SB-206553) and illustrate the use of aromatic disubstitution as a replacement for fused five-membered rings in the context of 5-HT2C/2B receptor antagonists. By targeting a region of space previously identified as sterically allowed at the 5-HT2C receptor but disallowed at the 5-HT2A receptor, we have identified a number of compounds which are the most potent and selective 5-HT2C/2B receptor antagonists yet reported. 46 (SB-221284) was selected on the basis of its overall biological profile for further evaluation as a novel, potential nonsedating anxiolytic agent. A CoMFA analysis of these compounds produced a model with good predictive value and in addition good qualitative agreement with both our 5-HT2C receptor model and our proposed binding mode for this class of ligands within that model.
5-HT1 receptors are members of the G-protein-coupled receptor superfamily and are negatively linked to adenylyl cyclase activity. The human 5-HT1B and 5-HT1D receptors (previously known as 5-HT1Dbeta and 5-HT1Dalpha, respectively), although encoded by two distinct genes, are structurally very similar. Pharmacologically, these two receptors have been differentiated using nonselective chemical tools such as ketanserin and ritanserin, but the absence of truly selective agents has meant that the precise function of the 5-HT1B and 5-HT1D receptors has not been defined. In this paper we describe how, using computational chemistry models as a guide, the nonselective 5-HT1B/5-HT1D receptor antagonist 4 was structurally modified to produce the selective 5-HT1B receptor inverse agonist 5, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6, 7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289). This compound is a potent antagonist of terminal 5-HT autoreceptor function both in vitro and in vivo.
Voltage‐sensitive Ca2+ channels (VSCCs) are often heteromultimeric complexes. The VSCC subtype specifically expressed by skeletal muscle has long been known to contain a γ subunit, γ1, that is only expressed in this tissue. Recent work, initiated by the identification of the mutation present in the stargazer mouse, has led to the identification of a series of novel potential Ca2+ channel γ subunits expressed in the CNS.
Based on bioinformatic techniques we identified and cloned the human γ2, γ3 and γ4 subunits.
TaqMan analysis was used to quantitatively characterise the mRNA expression patterns of all the γ subunits. All three subunits were extensively expressed in adult brain with overlapping but subunit‐specific distributions. γ2 and γ3 were almost entirely restricted to the brain, but γ4 expression was seen in a broad range of peripheral tissues.
Using a myc epitope the γ2 subunit was tagged both intracellularly at the C‐terminus and on a predicted extracellular site between the first and second transmembrane domains. The cellular distribution was then examined immunocytochemically, which indicated that a substantial proportion of the cellular pool of the γ2 subunit was present on the plasma membrane and provided initial evidence for the predicted transmembrane topology of the γ subunits.
Using co‐transfection techniques we investigated the functional effects of each of the γ subunits on the biophysics of the T‐type VSCC encoded by the α1I subunit. This revealed a substantially slowed rate of deactivation in the presence of γ2. In contrast, there was no significant corresponding effect of either γ3 or γ4 on α1I subunit‐mediated currents.
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