Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: A quantitative approach

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na+-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the α, β, and γ ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of α, β, and γ ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (β R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin

Pain differs from other sensations in many respects. Primary pain-sensitive neurons respond to a wide variety of noxious stimuli, in contrast to the relatively specific responses characteristic of other sensory systems, and the response is often observed to sensitize on repeated presentation of a painful stimulus, while adaptation is typically observed in other sensory systems. In most cases the cellular mechanisms of transduction and sensitization in response to painful stimuli are not understood. We report here that application of pulses of noxious heat to a subpopulation of isolated primary sensory neurons rapidly activates an inward current. The ion channel activated by heat discriminates poorly among alkali cations. Calcium ions both carry current and partially suppress the current carried by other ions. The current is markedly increased by bradykinin, a potent algogenic nonapeptide that is known to be released in vivo by tissue damage. Phosphatase inhibitors prolong the sensitization caused by bradykinin, and a similar sensitization is caused by activators of protein kinase C. We conclude that bradykinin sensitizes the response to heat by activating protein kinase C.

Ion channels gated by heat

All animals need to sense temperature to avoid hostile environments and to regulate their internal homeostasis. A particularly obvious example is that animals need to avoid damagingly hot stimuli. The mechanisms by which temperature is sensed have until recently been mysterious, but in the last couple of years, we have begun to understand how noxious thermal stimuli are detected by sensory neurons. Heat has been found to open a nonselective cation channel in primary sensory neurons, probably by a direct action. In a separate study, an ion channel gated by capsaicin, the active ingredient of chili peppers, was cloned from sensory neurons. This channel (vanilloid receptor subtype 1, VR1) is gated by heat in a manner similar to the native heat-activated channel, and our current best guess is that this channel is the molecular substrate for the detection of painful heat. Both the heat channel and VR1 are modulated in interesting ways. The response of the heat channel is potentiated by phosphorylation by protein kinase C, whereas VR1 is potentiated by externally applied protons. Protein kinase C is known to be activated by a variety of inflammatory mediators, including bradykinin, whereas extracellular acidification is characteristically produced by anoxia and inflammation. Both modulatory pathways are likely, therefore, to have important physiological correlates in terms of the enhanced pain (hyperalgesia) produced by tissue damage and inflammation. Future work should focus on establishing, in molecular terms, how a single ion channel can detect heat and how the detection threshold can be modulated by hyperalgesic stimuli.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.

At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation.

Ca(2+)-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine phosphatase.

Regulation of ion channel function by intracellular processes is fundamental for controlling synaptic signaling and integration in the nervous system. Currents mediated by N-methyl-D-aspartate (NMDA) receptors decline during whole-cell recordings and this may be prevented by ATP. We show here that phosphorylation is necessary to maintain NMDA currents and that the decline is not dependent upon Ca2+. A protein tyrosine phosphatase or a peptide inhibitor of protein tyrosine kinase applied intracellularly caused a decrease in NMDA currents even when ATP was included. On the other hand, pretreating the neurons with a membrane-permeant tyrosine kinase inhibitor occluded the decline in NMDA currents when ATP was omitted. In inside-out patches, applying a protein tyrosine phosphatase to the cytoplasmic face of the patch caused a decrease in probability of opening of NMDA channels. Conversely, open probability was increased by a protein tyrosine phosphatase inhibitor. These results indicate that NMDA channel activity is reduced by a protein tyrosine phosphatase associated with the channel complex.

Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.

Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Effects of Bisphenol A on ion channels: Experimental evidence and molecular mechanisms

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) produced in huge quantities in the manufacture of polycarbonate plastics and epoxy resins. It is present in most humans in developed countries, acting as a xenoestrogen and it is considered an environmental risk factor associated to several diseases. Among the whole array of identified mechanisms by which BPA can interfere with physiological processes in living organisms, changes on ion channel activity is one of the most poorly understood. There is still little evidence about BPA regulation of ion channel expression and function. However, this information is key to understand how BPA disrupts excitable and non-excitable cells, including neurons, endocrine cells and muscle cells. This report is the result of a comprehensive literature review on the effects of BPA on ion channels. We conclude that there is evidence to say that these important molecules may be key end-points for EDCs acting as xenoestrogens. However, more research on channel-mediated BPA effects is needed. Particularly, mechanistic studies to unravel the pathophysiological actions of BPA on ion channels at environmentally relevant doses.

Conotoxins as research tools and drug leads

The complex mixture of biologically active peptides that constitute the venom of Conus species provides a rich source of ion channel neurotoxins. These peptides, commonly known as conotoxins, exhibit a high degree of selectivity and potency for different ion channels and their subtypes making them invaluable tools for unravelling the secrets of the nervous system. Furthermore, several conotoxin molecules have profound applications in drug discovery, with some examples currently undergoing clinical trials. Despite their relatively easy access by chemical synthesis, rapid access to libraries of conotoxin analogues for use in structure-activity relationship studies still poses a significant limitation. This is exacerbated in conotoxins containing multiple disulfide bonds, which often require synthetic strategies utilising several steps. This review will examine the structure and activity of some of the known classes of conotoxins and will highlight their potential as neuropharmacological tools and as drug leads. Some of the classical and more recent approaches to the chemical synthesis of conotoxins, particularly with respect to the controlled formation of disulfide bonds will be discussed in detail. Finally, some examples of structure-activity relationship studies will be discussed, as well as some novel approaches for designing conotoxin analogues.

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an extemal I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

A CFTR chloride channel activator prevents HrpN(ea)-induced cell death in Arabidopsis thaliana suspension cells

Erwinia amylovora is a necrogenic bacterium that causes fire blight of the Maloideae subfamily of Roseacae, such as apple and pear. It provokes necrosis in aerial parts of susceptible host plants and the typical hypersensitive reaction in non-host plants. The secreted hatpin, HrpN(ea), is able by itself to induce an active cell death in non-host plants. Ion flux modulations were shown to be involved early in such processes but very few data are available on the plasma membrane ion channel activities responsible for the pathogen-induced ion fluxes. We show here that HrpNea induces cell death in non-host Arabidopsis thaliana suspension cells. We further show that two cystic fibrosis transmembrane conductance regulator modulators, glibenclamide and bromotetramisole, can regulate anion channel activities and HrpN(ea)-induced cell death. (c) 2005 Elsevier SAS. All rights reserved.

Conformational changes involved in MscL channel gating measured using FRET spectroscopy

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 angstrom upon channel activation.

IUPHAR-DB:the IUPHAR database of G protein-coupled receptors and ion channels

The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org. © 2008 The Author(s).