Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2018 May 15;135:e0196894. doi: 10.1371/journal.pone.0196894.
Show Gene links
Show Anatomy links
Molecular basis of inhibition of acid sensing ion channel 1A by diminazene.
Krauson AJ
,
Rooney JG
,
Carattino MD
.
???displayArticle.abstract???
Acid-sensing ion channels (ASICs) are trimeric proton-gated cation permeable ion channels expressed primarily in neurons. Here we employed site-directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASIC1a by diminazene. This compound inhibits mouse ASIC1a with a half-maximal inhibitory concentration (IC50) of 2.4 μM. At first, we examined whether neutralizing mutations of Glu79 and Glu416 alter diminazene block. These residues form a hexagonal array in the lower palm domain that was previously shown to contribute to pore opening in response to extracellular acidification. Significantly, single Gln substitutions at positions 79 and 416 in ASIC1a reduced diminazene apparent affinity by 6-7 fold. This result suggests that diminazene inhibits ASIC1a in part by limiting conformational rearrangement in the lower palm domain. Because diminazene is charged at physiological pHs, we assessed whether it inhibits ASIC1a by blocking the ion channel pore. Consistent with the notion that diminazene binds to a site within the membrane electric field, diminazene block showed a strong dependence with the membrane potential. Moreover, a Gly to Ala mutation at position 438, in the ion conduction pathway of ASIC1a, increased diminazene IC50 by one order of magnitude and eliminated the voltage dependence of block. Taken together, our results indicate that the inhibition of ASIC1a by diminazene involves both allosteric modulation and blocking of ion flow through the conduction pathway. Our findings provide a foundation for the development of more selective and potent ASIC pore blockers.
???displayArticle.pubmedLink???
29782492
???displayArticle.pmcLink???PMC5962070 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Fig 1. Diminazene is protonated at physiological pHs.(A) Chemical structure of diminazene. (B) Representative absorbance spectrums of diminazene in sodium phosphate solutions of pH 1, 7 and 13. Buffered solutions prepared as described in Materials and Methods contained 20 μM diminazene. The wavelength of maximum absorbance for diminazene at pH 7 and 13 were 370 nm and 425 nm, respectively. (C) Diminazene absorption as function of pH. Absorbance at 370 nm was normalized to the absorbance value of the diminazene solution at pH 8 (black dots, left y-axis). Absorbance at 425 nm was normalized to the absorbance value of the diminazene solution of pH 13 (red dots, right y-axis). Data are representative of two experiments.
Fig 2. Neutralization of Glu79 and Glu416 increases diminazene half-maximal inhibitory concentration.(A) Cartoon representation of chicken ASIC1 (cASIC1) in the desensitized state (PBD 4NYK). Inset, Close up view of residues Glu79 and Glu416 in the palm domain. (B) Representative tracing showing the inhibitory effect of diminazene on ASIC1a. Whole-cell currents were evoked by a change in extracellular pH from 8.0 to 6.5. (C) Time constant of desensitization (tau) for ASIC1a in the absence and presence of increasing concentrations of diminazene. Statistically significant differences in time constants of desensitization of ASIC1a before (control) and after diminazene are indicated as * p<0.05 and ** p<0.001 (n = 11–39, Kruskal-Wallis test followed by Dunn’s multiple comparisons test). (D) Neutralization of Glu79 and Glu416 in ASIC1a increases diminazene IC50. Whole-cell currents were evoked by a change in extracellular pH from 8.0 to solutions of lower pH. pH of activation was 6.5 for wild type ASIC1a and 5 for E79Q and E416Q. The relative response represents the ratio of the pH-elicited peak current after diminazene to the pH-elicited peak current before diminazene. Diminazene IC50 for ASIC1a was 2.42 μM (CI 1.56–3.75 μM, n = 10–14), for E79Q was 18.42 μM (CI 8.78–49.75 μM, n = 9–10) and for E416Q was 13.89 μM (CI 5.44–38.23 μM, n = 9–13).
Fig 3. Neutral substitutions at the acidic pocket do not alter diminazene block.(A) Cartoon representation of the acidic pocket of cASIC1 (PBD 4NYK). (B) Diminazene dose response curves for wild type ASIC1a and the 6NQ (E219Q/D237N/E238Q/D345N/D349N/D407N) mutant. Whole-cell currents were evoked by a change in extracellular pH from 8.0 to 6.5. The relative response represents the ratio of the pH-elicited peak current after diminazene to the pH-elicited peak current before diminazene. Diminazene IC50 for ASIC1a was 2.42 μM (CI 1.56–3.75 μM, n = 10–14) and for the 6NQ mutant was 2.03 μM (CI 0.88–5.31 μM, n = 10–21).
Fig 4. Voltage-dependent inhibition of ASIC1a by diminazene.(A) Representative ramp tracings from an oocyte expressing ASIC1a before and after diminazene. Ramps from +60 to -140 mV with a duration of 500 msec were generated in the absence and presence of diminazene (1 nM and 1 μM). ASIC currents were evoked by changing the extracellular pH from 8 to 6.5. Note that the inhibition of ASIC1a by 1 μM diminazene displays strong voltage dependence. (B) Normalized proton-evoked currents from oocytes expressing ASIC1a in the absence and presence of diminazene. Proton-evoked currents were elicited as indicated above in B. Proton-evoked currents measured in the presence of diminazene were normalized to the proton-evoked current measured at -120 mV before diminazene (n = 7–22). (C) Diminazene IC50 at each voltage was calculated from dose-response curves. The calculated diminazene IC50 was 2.48 μM (CI 1.48–4.04 μM, n = 7–22) at—40 mV, 1.82 μM (CI 1.16–2.75 μM, n = 7–22) at—60 mV, 1.41 μM (CI 0.91–2.10 μM, n = 7–22) at -80 mV, 0.99 μM (CI 0.63–1.49 μM, n = 7–22) at -100 mV, and 0.78 μM (CI 0.52–1.13 μM, n = 7–22) at -120 mV. (D) Diminazene IC50 shows a strong dependence with the membrane potential. Log IC50 values (C) were plotted as a function of the membrane potential. Data represent log IC50 ± SEM. Data were fitted by linear regression (slope 0.0063 ± 0.0002, r2 = 0.997, p<0.0001).
Fig 5. Diminazene binds to the pore of ASIC1a.(A) Sequence alignment for residues in the pore of ENaC/Deg channels. Note that many of the pore lining residues are conserved among ENaC and ASIC subunits, including the putative amiloride binding site (Gly438 in ASIC1a). (B) Representative ramp tracings from an oocyte expressing the G438A mutant before and after diminazene. Ramps from +60 to -140 mV with a duration of 500 msec were generated in the absence and presence of diminazene (1 μM and 30 μM). ASIC currents were evoked by changing the extracellular pH from 8 to 6.5. (C) Normalized proton-evoked currents from oocytes expressing G438A channels in the absence and presence of diminazene. Proton-evoked currents measured in the presence of diminazene were normalized to the proton-evoked current measured at -120 mV in the absence of diminazene (n = 9–27). (D) Diminazene IC50 at each voltage was calculated from dose-response curves. The calculated diminazene IC50 for the G438A mutant was 29 μM (CI 16 to 55 μM, n = 9–27) at—40 mV, 34 μM (CI 20 to 60 μM, n = 9–27) at—60 mV, 40 μM (CI 24 to 74 μM, n = 9–27) at -80 mV, 51 μM (CI 30 to 98 μM, n = 9–27) at -100 mV, and 66 μM (CI 38 to 140 μM, n = 9–27) at -120 mV. (E) Ala mutation at position 438 abolishes voltage dependence of diminazene block. Log IC50 values (C) were plotted as a function of the membrane potential. Data represent log IC50 ± SEM. Data were fitted by linear regression (slope -0.0045 ± 0.0003, r2 = 0.987, p<0.001).
Akopian,
A new member of the acid-sensing ion channel family.
2000, Pubmed
Akopian,
A new member of the acid-sensing ion channel family.
2000,
Pubmed
Arias,
Amiloride is neuroprotective in an MPTP model of Parkinson's disease.
2008,
Pubmed
Babinski,
Molecular cloning and regional distribution of a human proton receptor subunit with biphasic functional properties.
1999,
Pubmed
,
Xenbase
Baconguis,
X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na(+)-selective channel.
2014,
Pubmed
Berdiev,
Acid-sensing ion channels in malignant gliomas.
2003,
Pubmed
Bubien,
Cation selectivity and inhibition of malignant glioma Na+ channels by Psalmotoxin 1.
2004,
Pubmed
Bässler,
Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b.
2001,
Pubmed
,
Xenbase
Carattino,
Contribution of residues in second transmembrane domain of ASIC1a protein to ion selectivity.
2012,
Pubmed
,
Xenbase
Chen,
A role for ASIC3 in the modulation of high-intensity pain stimuli.
2002,
Pubmed
Chen,
Diarylamidines: high potency inhibitors of acid-sensing ion channels.
2010,
Pubmed
,
Xenbase
Chen,
Interaction of acid-sensing ion channel (ASIC) 1 with the tarantula toxin psalmotoxin 1 is state dependent.
2006,
Pubmed
,
Xenbase
Chen,
The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity.
2005,
Pubmed
,
Xenbase
Chen,
A sensory neuron-specific, proton-gated ion channel.
1998,
Pubmed
Dawson,
Structure of the acid-sensing ion channel 1 in complex with the gating modifier Psalmotoxin 1.
2012,
Pubmed
Della Vecchia,
Gating transitions in the palm domain of ASIC1a.
2013,
Pubmed
,
Xenbase
Diochot,
A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons.
2004,
Pubmed
,
Xenbase
Diochot,
Black mamba venom peptides target acid-sensing ion channels to abolish pain.
2012,
Pubmed
,
Xenbase
Dorofeeva,
Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneurons.
2008,
Pubmed
Dubé,
Electrophysiological and in vivo characterization of A-317567, a novel blocker of acid sensing ion channels.
2005,
Pubmed
Escoubas,
Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels.
2000,
Pubmed
,
Xenbase
Friese,
Acid-sensing ion channel-1 contributes to axonal degeneration in autoimmune inflammation of the central nervous system.
2007,
Pubmed
García-Añoveros,
BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels.
1997,
Pubmed
Gründer,
A new member of acid-sensing ion channels from pituitary gland.
2000,
Pubmed
,
Xenbase
Holland,
Acid-sensing ion channel 1: a novel therapeutic target for migraine with aura.
2012,
Pubmed
Kapoor,
Knockdown of ASIC1 and epithelial sodium channel subunits inhibits glioblastoma whole cell current and cell migration.
2009,
Pubmed
Kashlan,
On the interaction between amiloride and its putative alpha-subunit epithelial Na+ channel binding site.
2005,
Pubmed
,
Xenbase
Kellenberger,
Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.
2002,
Pubmed
Kellenberger,
International Union of Basic and Clinical Pharmacology. XCI. structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel.
2015,
Pubmed
Kellenberger,
Mutations in the epithelial Na+ channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate.
2003,
Pubmed
,
Xenbase
Krauson,
Independent contribution of extracellular proton binding sites to ASIC1a activation.
2013,
Pubmed
,
Xenbase
Kuduk,
Synthesis, structure-activity relationship, and pharmacological profile of analogs of the ASIC-3 inhibitor A-317567.
2010,
Pubmed
Leng,
Amiloride Analogs as ASIC1a Inhibitors.
2016,
Pubmed
Lingueglia,
Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel.
1995,
Pubmed
,
Xenbase
Palmer,
Voltage-dependent block by amiloride and other monovalent cations of apical Na channels in the toad urinary bladder.
1984,
Pubmed
Price,
Cloning and expression of a novel human brain Na+ channel.
1996,
Pubmed
,
Xenbase
Price,
The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.
2001,
Pubmed
Schild,
Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.
1997,
Pubmed
,
Xenbase
Schmidt,
Diminazene Is a Slow Pore Blocker of Acid-Sensing Ion Channel 1a (ASIC1a).
2017,
Pubmed
,
Xenbase
Snyder,
A pore segment in DEG/ENaC Na(+) channels.
1999,
Pubmed
Tolino,
Insights into the mechanism of pore opening of acid-sensing ion channel 1a.
2011,
Pubmed
,
Xenbase
Ugawa,
Nafamostat mesilate reversibly blocks acid-sensing ion channel currents.
2007,
Pubmed
,
Xenbase
Vila-Carriles,
Surface expression of ASIC2 inhibits the amiloride-sensitive current and migration of glioma cells.
2006,
Pubmed
Voilley,
Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors.
2001,
Pubmed
Waldmann,
The mammalian degenerin MDEG, an amiloride-sensitive cation channel activated by mutations causing neurodegeneration in Caenorhabditis elegans.
1996,
Pubmed
Wemmie,
The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory.
2002,
Pubmed
Wemmie,
Acid-sensing ion channels in pain and disease.
2013,
Pubmed
Wiemuth,
The pharmacological profile of brain liver intestine Na+ channel: inhibition by diarylamidines and activation by fenamates.
2011,
Pubmed
,
Xenbase
Wong,
Blocking acid-sensing ion channel 1 alleviates Huntington's disease pathology via an ubiquitin-proteasome system-dependent mechanism.
2008,
Pubmed
Xiong,
Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels.
2004,
Pubmed
Zha,
Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines.
2006,
Pubmed
de Weille,
Identification, functional expression and chromosomal localisation of a sustained human proton-gated cation channel.
1998,
Pubmed