Calcitonin-gene-related peptide (CGRP) and adrenomedullin are related peptides with distinct pharmacological profiles. Here we show that a receptor with seven transmembrane domains, the calcitonin-receptor-like receptor (CRLR), can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a new family of single-transmembrane-domain proteins, which we have called receptor-activity-modifying proteins or RAMPs, are expressed. RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents the receptor at the cell surface as a mature glycoprotein and a CGRP receptor. RAMP2-transported receptors are core-glycosylated and are adrenomedullin receptors.

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [IK(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of IK(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. IK(M) was identified, on the basis of biophysical and pharmacological properties, in cultured neurons isolated from dorsal root ganglia (DRGs) from 17-d-old rats. Currents were inhibited by the M-channel blockers linopirdine (IC50, 2.1 microm) and XE991 (IC50, 0.26 microm) and enhanced by retigabine (10 microm). The expression of neuronal KCNQ subunits in DRG neurons was confirmed using reverse transcription-PCR and single-cell PCR analysis and by immunofluorescence. Retigabine, applied to the dorsal spinal cord, inhibited C and Adelta fiber-mediated responses of dorsal horn neurons evoked by natural or electrical afferent stimulation and the progressive "windup" discharge with repetitive stimulation in normal rats and in rats subjected to spinal nerve ligation. Retigabine also inhibited responses to intrapaw application of carrageenan in a rat model of chronic pain; this was reversed by XE991. It is suggested that IK(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target.

Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1-) into COS-7 cells induced specific 125I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP-induced amylin receptors revealed two distinct phenotypes. The RAMP 1-derived amylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 +/- 1.44 x 10(-10) M), a high to moderate affinity for rat amylin (IC50, 7.86 +/- 4.49 x 10(-9) M) and human CGRPalpha (IC50, 2.09 +/- 1.63 x 10(-8) M), and a low affinity for human CT (IC50, 4.47 +/- 0.78 x 10(-7) M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPalpha was markedly reduced (IC50, 1.12 +/- 0.45 x 10(-7) M; P <.05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1- were reflective of the phenotypes seen in competition for amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)I1- induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins.

Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination in Xenopus oocytes. Application of 10 microM retigabine to oocytes expressing the KCNQ2/3 heteromeric channel shifted both the activation threshold and voltage for half-activation by approximately 20 mV in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between -80 mV and +20 mV. Retigabine also had a marked effect on KCNQ current kinetics, increasing the rate of channel activation but slowing deactivation at a given test potential. Similar effects of retigabine were observed in oocytes expressing KCNQ2 alone, suggesting that KCNQ2 may be the molecular target of retigabine. Membrane potential recordings in oocytes expressing the KCNQ2/3 heteromeric channel showed that application of retigabine leads to a concentration-dependent hyperpolarization of the oocyte, from a resting potential of -63 mV under control conditions to -85 mV in the presence of 100 microM retigabine (IC(50) = 5.2 microM). In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.

The calcitonin receptor-like receptor (CRLR) can function as either a receptor for calcitonin gene-related peptide (CGRP) or for adrenomedullin (ADM), depending upon the coexpression of a novel family of single transmembrane proteins, which we have called receptor activity modifying proteins or RAMPs. RAMPs 1, 2, and 3 transport CRLR to the plasma membrane with similar efficiencies, however RAMP1 presents CRLR as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor. Characterization of the RAMP2/CRLR and RAMP3/CRLR receptors in HEK293T cells by radioligand binding (125I-ADM as radioligand), functional assay (cAMP measurement), or biochemical analysis (SDS-polyacrylamide gel electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% identity. Chimeric proteins were created with the transmembrane and cytosolic portions of RAMP1 associated with the amino terminus of RAMP2 (RAMP2/1) and vice versa (RAMP1/2). Coexpression of RAMP2/1 with CRLR formed a core glycosylated ADM receptor, whereas the RAMP1/2 chimera generated both core glycosylated and mature forms of CRLR and enabled both ADM and CGRP receptor binding. Hence, the glycosylation state of CRLR appears to correlate with its pharmacology.

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SummaryVarious ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, invasion and metastasis. Expression of functional voltage-gated Na + channels (Na v ) is implicated in the metastatic potential of breast, prostate, lung and colon cancer cells. However, the cellular mechanisms that regulate Na v expression in cancer remain largely unknown. Growth factors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channel expression and activity in non-cancerous cells. Here, we examine the role of epidermal growth factor receptor (EGFR) signalling and Na v in non-small cell lung carcinoma (NSCLC) cell lines. We show unequivocally, that functional expression of the a subunit Na v 1.7 promotes invasion in H460 NSCLC cells. Inhibition of Na v 1.7 activity (using tetrodotoxin) or expression (by using small interfering RNA), reduces H460 cell invasion by up to 50%. Crucially, non-invasive wild type A549 cells lack functional Na v , whereas exogenous overexpression of the Na v 1.7 a subunit is sufficient to promote TTX-sensitive invasion of these cells. EGF/EGFR signalling enhances proliferation, migration and invasion of H460 cells but we find that, specifically, EGFR-mediated upregulation of Na v 1.7 is necessary for invasive behaviour in these cells. Examination of Na v 1.7 expression at mRNA, protein and functional levels further reveals that EGF/ EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of channel expression to promote cellular invasion. Immunohistochemistry of patient biopsies confirms the clinical relevance of Na v 1.7 expression in NSCLC. Thus, Na v 1.7 has significant potential as a new target for therapeutic intervention and/or as a diagnostic or prognostic marker in NSCLC.