Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R. J., Derkach, V. & Surprenant, A. (1992) Nature (London)

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

SUMMARYWe isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X 4 receptor (87% identity). By fluorescence in situ hybridization, the human P2X 4 gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X 4 transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X 4 receptor in Xenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embryonic kidney 293 cells transfected with human P2X 4 cDNA allowed us to determine the fraction of the current carried by Ca 2ϩ ; this was ϳ8%, demonstrating a high Ca 2ϩ permeability.Low extracellular Zn 2ϩ concentrations (5-10 M) increase the apparent gating efficiency of human P2X 4 by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (Ͼ100 M) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X 4 receptor displays a very similar agonist potency profile to that of rat P2X 4 (ATP Ͼ Ͼ 2-methylthio-ATP Ն CTP Ͼ ␣,␤-methylene-ATP Ͼ dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid.

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Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full-length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40-47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK-293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP-activated current reveals little agonist desensitization, is not activated by a,~meATP and is completely blocked by suramin and PPADS.