Article

Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature

Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA.
Nature (Impact Factor: 41.46). 10/2007; 449(7160):316-23. DOI: 10.1038/nature06163
Source: PubMed

ABSTRACT

Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and are implicated in perception of pain, ischaemic stroke, mechanosensation, learning and memory. Here we report the low-pH crystal structure of a chicken ASIC1 deletion mutant at 1.9 A resolution. Each subunit of the chalice-shaped homotrimer is composed of short amino and carboxy termini, two transmembrane helices, a bound chloride ion and a disulphide-rich, multidomain extracellular region enriched in acidic residues and carboxyl-carboxylate pairs within 3 A, suggesting that at least one carboxyl group bears a proton. Electrophysiological studies on aspartate-to-asparagine mutants confirm that these carboxyl-carboxylate pairs participate in proton sensing. Between the acidic residues and the transmembrane pore lies a disulphide-rich 'thumb' domain poised to couple the binding of protons to the opening of the ion channel, thus demonstrating that proton activation involves long-range conformational changes.

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    • "The structure of Deg/ENaC channels was revealed by the crystallization of chicken acid-sensing ion channel 1 (ASIC1; Baconguis and Gouaux 2012; Baconguis et al. 2013, 2014; Gonzales et al. 2009; Jasti et al. 2007). Deg/ ENaC channels are trimeric containing three similar subunits arranged to form a central conductive pore. "
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    ABSTRACT: These studies test whether three disease-causing mutations in genes (SCNN1A and SCNN1G) encoding subunits of the epithelial Na(+) channel, ENaC, affect the biophysical and gating properties of this important renal ion channel. The S562P missense mutation in αENaC and the K106_S108delinsN mutation in γENaC are associated with pseudohypoaldosteronism type 1 (PHA1). The N530S missense mutation in γENaC causes Liddle's syndrome. Incorporation of S562P into αENaC and K106_S108N into γENaC resulted in significant decreases in macroscopic ENaC currents. Conversely, incorporation of N530S into γENaC increased macroscopic ENaC current. The S562P substitution resulted in a nonfunctional channel. The K106_S108N mutation produced a functional channel having a normal macroscopic current-voltage relation, there was a slight but significant decrease in unitary conductance and a marked decrease in single-channel open probability. The N530S substitution increased single-channel open probability having no effect on the macroscopic current-voltage relation or unitary conductance of the channel. These findings are consistent with mutation of residues at 562 in αENaC and 530 in γENaC, and a 3' splice site in SCNN1G (318-1 G→A; K106_108SdelinsN) resulting in aberrant ENaC activity due to changes in the biophysical and gating properties of the channel. Such changes likely contribute to the cellular mechanism underpinning the PHA1 and Liddle's syndrome caused by these mutations in ENaC subunits.
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    • "Abbreviations include CRD, TM, and conserved amino acid residues motifs. Crystal structure of ASIC1 is from the Protein Data Bases (see legend of Fig. 1 for database references) doi: 10.2210/PBD:ID 2qts (Jasti et al. 2007). (C) Alignment of conserved pore region in SCNN1/ASIC-like channels. "
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    ABSTRACT: Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian "synaptic" and "neuronal" protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true "pan-synaptic" genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores-the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of "synaptic" proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of transcriptomes from 10 different ctenophores did not detect recognized orthologs of synthetic enzymes encoding several classical, low-molecular-weight (neuro)transmitters; glutamate signaling machinery is one of the few exceptions. Novel peptidergic signaling molecules were predicted for ctenophores, together with the diversity of putative receptors including SCNN1/amiloride-sensitive sodium channel-like channels, many of which could be examples of a lineage-specific expansion within this group. In summary, our analysis supports the hypothesis of independent evolution of neurons and, as corollary, a parallel evolution of synapses. We suggest that the formation of synaptic machinery might occur more than once over 600 million years of animal evolution.
    Full-text · Article · Oct 2015 · Integrative and Comparative Biology
    • "The " classical " ENaC consists of three homologous subunits (a, b and g) [4] which likely assemble as a heterotrimer [5] and build a sodium-selective pore in the plasma membrane. In primates, there is a fourth subunit (d) which can replace the a-subunit and build a functional channel together with the b-and g-subunits [6]. "
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    ABSTRACT: The Epithelial Sodium Channel (ENaC) is a heterotrimeric ion channel which can be either formed by assembly of its α-, β- and γ-subunits or, alternatively, its δ-, β- and γ-subunits. The physiological function of αβγ-ENaC is well established, but the function of δβγ-ENaC remains elusive. The azo-dye Evans Blue (EvB) has been routinely used to discriminate between the two channel isoforms by decreasing transmembrane currents and amiloride-sensitive current fractions of δβγ-ENaC expressing Xenopus oocytes. Even though these results could be reproduced, it was found by precipitation experiments and spectroscopic methods that the cationic amiloride and the anionic EvB directly interact in solution, forming a strong complex. Thereby a large amount of pharmacologically available amiloride is removed from physiological buffer solutions and the effective amiloride concentration is reduced. This interaction did not occur in the presence of albumin. In microelectrode recordings, EvB was able to abrogate the block of δβγ-ENaC by amiloride or its derivative benzamil. In sum, EvB reduces amiloride-sensitive ion current fractions in electrophysiological experiments. This is not a result of a specific inhibition of δβγ-ENaC but rather represents a pharmacological artefact. EvB should therefore not be used as an inhibitor of δ-ENaC.
    No preview · Article · Sep 2015 · Biochemical and Biophysical Research Communications
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