Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca2+‐dependent Cl− currents. The present study examines the relationship between the hClCa1 protein and Ca2+‐dependent Cl− currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl− currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca2+‐dependent Cl− currents and show that hClCa1 increases the amplitude of Ca2+‐dependent Cl− currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl− conductance while decreasing the GSCN−/GCl− conductance ratio from ∼2–3 to ∼1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca2+‐dependent Cl− channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca2+‐dependent Cl− channels by lowering the energy barriers for ion translocation through the pore.

Ion channels are cellular proteins that conduct the movement of ions from one side of a membrane to the other. The resultant changes in local ion concentrations and electrical field play pivotal roles in physiological processes, as wide ranging as cell to cell communication, cell proliferation and secretion. This chapter provides a brief historical perspective then introduces the basic theory, terminology, and generic structural and functional features of ion channels. In addition, an overview of relevant ion channel methodologies is provided. The chapter aims to set the scene and to equip the non-specialist reader with sufficient background and understanding to comprehend and enjoy subsequent chapters which provide a more detailed analysis of channel families and individual channels.

The ability to apply large-scale screening formats to measures of ion channel function offers immense opportunities for drug discovery and academic research. Technologies have been developed over the last several years that now provide the ability to screen large numbers of compounds and natural products on ion channel function to find novel drugs. Application of these technologies has vastly improved the capabilities of ion channel drug discovery and provides an avenue to accelerate discoveries of ion channel biology. These advances have largely arisen from the development and application of instruments and reporters of membrane potential and ion movements in cells used to measure functional activity of ion channels. This article endeavors to describe the practical applications of these technologies in developing, validating, and implementing high throughput screening assay formats to different types of ion channels.

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.

Conference Article| August 01 1999 The N-terminus of RAMPs is a critical determinant of the glycosylation state and ligand binding of calcitonin receptor-like receptor S. M. Foord; S. M. Foord 1Receptor Systems, Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. Search for other works by this author on: This Site PubMed Google Scholar A. Wise; A. Wise 1Receptor Systems, Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. Search for other works by this author on: This Site PubMed Google Scholar J. Brown; J. Brown 1Receptor Systems, Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. Search for other works by this author on: This Site PubMed Google Scholar M. J. Main; M. J. Main 1Receptor Systems, Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. Search for other works by this author on: This Site PubMed Google Scholar N. J. Fraser N. J. Fraser 1Receptor Systems, Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1999) 27 (4): 535–539. https://doi.org/10.1042/bst0270535 Article history Received: March 05 1999 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation S. M. Foord, A. Wise, J. Brown, M. J. Main, N. J. Fraser; The N-terminus of RAMPs is a critical determinant of the glycosylation state and ligand binding of calcitonin receptor-like receptor. Biochem Soc Trans 1 August 1999; 27 (4): 535–539. doi: https://doi.org/10.1042/bst0270535 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CGRP, calcitonin gene-related peptide, ADM, adrenomedullin, CRLR, calcitonin receptor-like receptor, CFTR, cystic fibrosis transmembrane regulator, ER, endoplasmic reticulum, RAMP, receptor activity modifying protein This content is only available as a PDF. © 1999 Biochemical Society1999 Article PDF first page preview Close Modal You do not currently have access to this content.

The inwardly rectifying potassium channel Kir2.3 possesses extracellular cysteine residues at positions 113, 140 and 145, as well as at position 79 near the outer membrane boundary. In this study, we have investigated the roles of these extracellular cysteine residues in mediating inhibition of the Kir2.3 channel by the cysteine-reactive reagents para-chloromercuribenzenesulphonate (PCMBS) and thimerosal, and the oxidizing agent hydrogen peroxide (H2O2). We have also compared the effects of these reagents with those on Kir1.1 channels (which do not possess cysteine residues equivalent to 140 and 79 in Kir2.3 channels). Mutant channels were made in which cysteine residues were mutated to serine by site-directed mutagenesis. Wild-type or mutant cRNA was injected into Xenopus oocytes and voltage-clamp recordings made 1–2 days later. Wild-type Kir2.3 currents were significantly inhibited by PCMBS, thimerosal and H2O2. Currents for mutants Kir2.3 C79S and C140S were also inhibited by PCMBS, thimerosal and H2O2. These mutations affected the time course of inhibition by all three reagents. For PCMBS, a slow component of inhibition was absent for the C79S mutation, and a fast component was absent for C140S. For the double mutation C79S/C140S, PCMBS no longer had any effect. For thimerosal, there was a slower time course for C140S, a faster time course for C79S, and a delayed onset for C79S/C140S. For H2O2, the main effect was a delayed onset for the double mutant. The reducing agent dithiothreitol (DTT) reversed the inhibition by both PCMBS and thimerosal of wild-type and mutant currents, but not the inhibition due to H2O2. Finally, wild-type Kir1.1 currents were not significantly inhibited by the application of either PCMBS or thimerosal, while H2O2 produced small inhibition. The results taken together indicate that inhibition by the cysteine-reactive reagent PCMBS is mediated through cysteine residues 79 and 140 in Kir2.3 channels, with C79 mediating a slow component of inhibition and C140 a faster component, and that both residues are extracellularly exposed. The data indicate that these two cysteine residues are also main sites for inhibition by thimerosal and H2O2 but, unlike for PCMBS, an additional non-extracellular inhibitory site(s) must also be involved.

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.

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes. The discovery of the heterodimeric nature of the GABA(B) receptor has already changed the way we think about GPCR function and it is likely that future studies will change our understanding about how receptor subtypes can be formed.

The GABA(B) receptor has been shown to consist of a heterodimer of two related 7-transmembrane receptors GABAB-R1 and GABA(B)-R2. These receptors share close homology to the Ca2+-sensing receptor and also to the metabotropic glutamate receptors, which have also been shown to respond to extracellular calcium. We show here that the GABA(B) receptor also has Ca2+ sensing properties. Ca2+ (0.001-1 mM) potentiated the GABA stimulation of [35S]GTPgammaS binding in membranes prepared from CHO cells stably expressing the GABA(B)-R1/R2 heterodimer. The GABA EC50 was reduced from 72 to 7.7 microM by addition of 1 mM Ca2+, with no change in the maximum response. A similar effect was observed in membranes from rat brain cortex. Ca2+ also potentiated GABA inhibition of forskolin-stimulated cAMP levels in the CHO cells and enhanced coupling to GIRK K+ channels in Xenopus oocytes. Other divalent cations were ineffective. The effects of Ca2+ were found to be agonist dependent with baclofen having a reduced sensitivity compared to GABA. Calcium appears to act allosterically to enhance GABA responses at the GABA(B) receptor, however, unlike the Ca2+-sensing receptor and some of the mGluR family, Ca2+ does not act as a ligand in its own right.

Swiss 3T3 cell lines were constructed co-expressing receptor activity modifying protein (RAMP) 1 with the calcitonin receptor-like receptor (CRLR), and showed 125I-calcitonin-gene-related peptide (CGRP) 1 binding indicative of a type I CGRP receptor. Application of CGRP1 led to an increase in cAMP, which in 2/5 cell lines was augmented following pertussis toxin (PTX) pre-treatment. In Xenopus oocytes, expression of RAMP1, which potentiates an endogenous CGRP receptor, led to constitutive activation of co-expressed GIRK potassium channels. This potassium current was increased following CGRP application or co-expression of CRLR, but decreased by PTX or co-expression of transducin. We conclude that the CGRP receptor can signal to both PTX sensitive and insensitive G proteins.