Guy Champigny

Molecular cloning of a non-inactivating proton-gated Na^+ channel specific for sensory neurons

We have cloned and expressed a novel proton-gated Na+ channel subunit that is specific for sensory neurons. In COS cells, it forms a Na+ channel that responds to a drop of the extracellular pH with both a rapidly inactivating and a sustained Na+ current. This biphasic kinetic closely resembles that of the H+-gated current described in sensory neurons of dorsal root ganglia (1). Both the abundance of this novel H+-gated Na+ channel subunit in sensory neurons and the kinetics of the channel suggest that it is part of the channel complex responsible for the sustained H+-activated cation current in sensory neurons that is thought to be important for the prolonged perception of pain that accompanies tissue acidosis (1, 2).

Isolation of a Tarantula Toxin Specific for a Class of Proton-gated Na+ Channels

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H+-gated Na+ channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC50 = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties.

Proton-gated cation channels are acid sensors that are present in both sensory neurons and in neurons of the central nervous system. One of these acid-sensing ion channels (ASIC) has been recently cloned. This paper shows that ASIC and the mammalian degenerin MDEG, which are colocalized in the same brain regions, can directly associate with each other. Immunoprecipitation of MDEG causes coprecipitation of ASIC. Moreover, coexpression of ASIC and MDEG subunits in Xenopus oocytes generates an amiloride-sensitive H+-gated Na+ channel with novel properties (different kinetics, ionic selectivity, and pH sensitivity). In addition, coexpression of MDEG with mutants of the ASIC subunit can create constitutively active channels that become completely nonselective for Na+ versusK+ and H+-gated channels that have a drastically altered pH sensitivity compared with MDEG. These data clearly show that ASIC and MDEG can form heteromultimeric assemblies with novel properties. Heteromultimeric assembly is probably used for creating a diversity of H+-gated cation channels acting as neuronal acid sensors in different pH ranges.

H+‐Gated Cation Channelsa

ABSTRACT: H+‐gated cation channels are members of a new family of ionic channels, which includes the epithelial Na+ channel and the FMRFamide‐activated Na+ channel. ASIC, the first member of the H+‐gated Na+ channel subfamily, is expressed in brain and dorsal root ganglion cells (DRGs). It is activated by pHe variations below pH 7. The presence of this channel throughout the brain suggests that the H+ might play an essential role as a neurotransmitter or neuromodulator. The ASIC channel is also present in dorsal root ganglion cells, as is its homolog DRASIC, which is specifically present in DRGs and absent in the brain. Since external acidification is a major factor in pain associated with inflammation, hematomas, cardiac or muscle ischemia, or cancer, these two channel proteins are potentially central players in pain perception. ASIC activates and inactivates rapidly, while DRASIC has both a fast and sustained component. Other members of this family such as MDEG1 and MDEG2 are either H+‐gated Na+ channels by themselves (MDEG1) or modulators of H+‐gated channels formed by ASIC and DRASIC. MDEG1 is of particular interest because the same mutations that produce selective neurodegeneration in C. elegans mechanosensitive neurons, when introduced in MDEG1, also produce neurodegeneration. MDEG2 is selectively expressed in DRGs, where it assembles with DRASIC to radically change its biophysical properties, making it similar to the native H+‐gated channel, which is presently the best candidate for pain perception.

Zn2+ and H+ Are Coactivators of Acid-sensing Ion Channels

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in sensory neurons, where they are thought to be involved in pain perception associated with tissue acidosis. They are also expressed in brain. A number of brain regions, like the hippocampus, contain large amounts of chelatable vesicular Zn2+. This paper shows that Zn2+ potentiates the acid activation of homomeric and heteromeric ASIC2a-containing channels (i.e.ASIC2a, ASIC1a+2a, ASIC2a+3), but not of homomeric ASIC1a and ASIC3. The EC50 for Zn2+ potentiation is 120 and 111 μm for the ASIC2a and ASIC1a+2a current, respectively. Zn2+ shifts the pH dependence of activation of the ASIC1a+2a current from a pH0.5 of 5.5 to 6.0. Systematic mutagenesis of the 10 extracellular histidines of ASIC2a leads to the identification of two residues (His-162 and His-339) that are essential for the Zn2+ potentiating effect. Mutation of another histidine residue, His-72, abolishes the pH sensitivity of ASIC2a. This residue, which is located just after the first transmembrane domain, seems to be an essential component of the extracellular pH sensor of ASIC2a.

Genotype–phenotype analysis of a newly discovered family with Liddle's syndrome

Objective To investigate the clinical, biologic, and molecular abnormalities in a family with Liddles syndrome and analyze the short- and long-term efficacies of amiloride treatment. Patients The pedigree consisted of one affected mother and four children, of whom three suffered from earlyonset and moderate-to-severe hypertension. Methods In addition to the biochemical and hormonal measurements, genetic analysis of the carboxy terminus of the β subunit of the epithelial sodium channel (βENaC) was conducted through single-strand conformation analysis and direct sequencing. The functional properties of the mutation were analyzed using the Xenopus expression system and compared with one mutation affecting the proline-rich sequence of the βENaC. Results Mild hypokalemia and suppressed levels of plasma renin and aldosterone were observed in all affected subjects. Administration of 10mg/day amiloride for 2 months normalized the blood pressure and plasma potassium levels of all of the affected subjects, whereas their plasma and urinary aldosterone levels remained surprisingly low. A similar pattern was observed after 11 years of follow-up, but a fivefold increase in plasma aldosterone was observed under treatment with 20mg/day amiloride for 2 weeks. Genetic analysis of the βENaC revealed a deletion of 32 nucleotides that had modified the open reading frame and introduced a stop codon at position 582. Expression of this β579del32 mutant caused a 3.7 ± 0.3-fold increase in the amiloridesensitive sodium current, without modification of the unitary properties of the channel. A similar increase was elicited by one mutation affecting the carboxy terminus of the βENaC. Conclusions This new mutation leading to Liddles syndrome highlights the importance of the carboxy terminus of the βENaC in the activity of the epithelial sodium channel. Small doses of amiloride are able to control the blood pressure on a long-term basis in this monogenic from of hypertension.

Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals

1 A degenerate polymerase chain reaction (PCR) homology screening procedure was applied to rat brain cDNA in order to identify novel genes belonging to the amiloride‐sensitive Na+ channel and degenerin (NaC/DEG) family of ion channels. A single gene was identified that encodes a protein related to but clearly different from the already cloned members of the family (18‐30 % amino acid sequence identity). Phylogenetic analysis linked this protein to the group of ligand‐gated channels that includes the mammalian acid‐sensing ion channels and the Phe‐Met‐Arg‐Phe‐amide (FMRFamide)‐activated Na+ channel. 2 Expression of gain‐of‐function mutants after cRNA injection into Xenopus laevis oocytes or transient transfection of COS cells induced large constitutive currents. The activated channel was amiloride sensitive (IC50, 1.31 μm) and displayed a low conductance (9‐10 pS) and a high selectivity for Na+ over K+ (ratio of the respective permeabilities, PNa+/PK+≥ 10), all of which are characteristic of NaC/DEG channel behaviour. 3 Northern blot and reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis revealed a predominant expression of its mRNA in the small intestine, the liver (including hepatocytes) and the brain. This channel has been called the brain‐liver‐intestine amiloride‐sensitive Na+ channel (BLINaC). 4 Corresponding gain‐of‐function mutations in Caenorhabditis elegans degenerins are responsible for inherited neurodegeneration in the nematode. Besides the BLINaC physiological function that remains to be established, mutations in this novel mammalian degenerin‐like channel might be of pathophysiological importance in inherited neurodegeneration and liver or intestinal pathologies.

The amiloride-sensitive Na+ channel: from primary structure to function.

Three homologous subunits of the amiloride-sensitive Na+ channel, entitled alpha, beta, and gamma, have been cloned either from distal colon of a steroid-treated rat or from human lung. The alpha, beta, and gamma subunits have similarities with degenerins, a family of proteins found in the mechanosensory neurons of the nematode Caenorhabditis elegans. All these proteins are characterized by the presence of a large extracellular domain, located between two transmembrane alpha-helices, and by short NH2 and COOH terminal cytoplasmic segments. They constitute the first members of a new gene super-family of ionic channels. The epithelial Na+ channel is specifically expressed at the apical membrane of Na(+)-reabsorbing epithelial cells. Its activity is controlled by several distinct hormones, especially by corticosteroids. These hormones act either transcriptionally (such as aldosterone in distal colon, or glucocorticoids in lung) and/or post-transcriptionally (such as aldosterone in kidney). Recent works have provided new insights in the function of that important osmoregulatory system.

Ca2+ channel blockers inhibit secretory Cl− channels in intestinal epithelial cells

Outwardly rectifying Cl- channels are present in the human colonic cell line (HT29D4). The classical Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoate inhibits Cl- channel activity with a K0.5 value of 20 microM. Epithelial Cl- channel activity is inhibited by Ca2+ channel blockers. Phenylalkylamines are the most effective inhibitors. (+/-)Verapamil and (-)desmethoxyverapamil induce flickering and then the complete blockade of Cl- channels recorded from outside-out patches. K0.5 values are 60 microM and 100 microM for (-)desmethoxyverapamil and (+/-)verapamil, respectively. Other classes of L-type Ca2+ channel blockers have also been studied but they are less active.

Lack of effect of Presenilin 1, βAPP and their Alzheimer's disease-related mutated forms on Xenopus oocytes membrane currents

The effect of the microinjection of Xenopus oocytes with various cRNAs coding for Presenilin 1 and four mutated presenilins linked to early onset familial forms of Alzheimers disease was examined. These cRNAs were injected either alone or in combination with the cRNA encoding betaAPP751 and the Swedish mutated form of betaAPP751 known to produce exacerbated amount of Abeta. Current-voltage relationships generated by voltage step were recorded. None of the cRNA injected alone or in combination displayed the ability to modify the current recorded with naive cells. Altogether, this study shows that Presenilin 1 does not mediate membrane currents and is more likely involved in the physiopathological maturation of betaAPP.