Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

Zha, Xiang-ming; Wang, Runping; Collier, Dan M.; Snyder, Peter M.; Wemmie, John A.; Welsh, Michael J.

doi:10.1073/pnas.0813402106

Cited by 57 publications

(69 citation statements)

References 36 publications

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“…We performed Western blot analysis under reducing and non-reducing conditions with the pool of Y424C-G428C expressed at the cell surface to determine the presence of intersubunit disulfide bonds. Oocytes were homogenized in the presence of NEM to prevent the formation of intersubunit disulfide bonds between intracellular Cys residues (15). A band of ϳ60 kDa, consistent with the molecular mass of a monomer, was immunodetected under reducing and non-reducing conditions in the surface pool recovered from oocytes expressing G428C-HA (Fig.…”

Section: Resultsmentioning

confidence: 99%

Insights into the Mechanism of Pore Opening of Acid-sensing Ion Channel 1A

Tolino

Okumura

Kashlan

et al. 2011

Journal of Biological Chemistry

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Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains. Acid-sensing ion channels (ASICs)2 are voltage-independent ligand-gated ion channels expressed throughout neurons of the mammalian central and peripheral nervous systems (1, 2). ASICs are members of the Epithelial Sodium Channel/Degenerin (ENaC/Deg) family, a large group of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume, and airway surface liquid volume (2-5). These channels are organized as homo-or hetero-trimers. Each subunit has two transmembrane segments (TMs) connected by a large extracellular region with the N and C termini on the intracellular side (6, 7). ASICs are constitutively closed and undergo activation and desensitization in response to extracellular acidification. Seven ASIC subunits are expressed in mammals: ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3, and ASIC4 (2). The sensitivity of activation and desensitization by protons depend on the specific subunits forming the channel complex.The pore of ASIC1 in the presumed desensitized-like state has an hourglass-like shape defined mainly by residues from TM2, although residues from TM1 form a portion of the outer vestibule (6, 7). The TM2 helices are tilted by a ϳ50°angle from the membrane normal, and their intersection likely constitutes the desensitization gate. The TM1 segments are in contact with the lipid bilayer and with the TM2 segments in their own and neighboring subunits. Compelling evidence suggests that ASIC activation involves protonation of multiple residues within the extracellular region of the protein (6, 8 -10). A fundamental question regarding the mechanism of gating of ENaC/Deg channels is how extracellular cues trigger pore opening and closing events. The extracellular region of ASIC1 is organized in discrete subdomains referred to as the palm, ...

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Section: Resultsmentioning

confidence: 99%

Insights into the Mechanism of Pore Opening of Acid-sensing Ion Channel 1A

Tolino

Okumura

Kashlan

et al. 2011

Journal of Biological Chemistry

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“…Surface Biotinylation and NeutrAvidin Pulldown Assay, followed by Western Blotting-Surface expression of the D238A/ E239A/D260A triple mutant of cASIC1a was determined using surface biotinylation and NeutrAvidin pulldown, followed by Western blotting as described by Zha et al (23). The triple mutant with a FLAG tag introduced at the N terminus expressed in HEK293T cells was used for these experiments.…”

Section: Methodsmentioning

confidence: 99%

Proton-mediated Conformational Changes in an Acid-sensing Ion Channel

Ramaswamy

MacLean

Gorfe

et al. 2013

Journal of Biological Chemistry

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Background: Protons activate acid-sensing ion channels (ASICs). Results: Proton binding leads to a movement involving the thumb and finger subdomains. Mutation of carboxylates lining the finger leads to loss of activation and loss of this movement. Conclusion:The carboxylates lining the finger domain are essential for the movement of the thumb and finger domains and in activation. Significance: This study provides insight into proton-induced conformational changes in ASICs.

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“…In contrary, H 2 O 2 treatment actually reduced the proportion of surface ASIC1a channels and therefore suppressed H þ -induced currents. 24 The above data thus suggest that ASIC1a proteins participate in H 2 O 2 -induced neuronal death but in a manner that is independent of their plasma membrane ASIC1a channels.…”

Section: Resultsmentioning

confidence: 83%

Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death

et al. 2013

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Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a À / À mice exhibit significantly enhanced Ca 2 þ retention capacity and accelerated Ca 2 þ uptake rate. When challenged with hydrogen peroxide (H 2 O 2 ), ASIC1a À / À neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H 2 O 2 -induced neuronal death, which is MPT dependent, is reduced in ASIC1a À / À neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a À / À mouse brain, which also displays marked changes (42-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death. The acid-sensing ion channels (ASICs) represent a subfamily of degenerin/epithelial Na þ channels that are activated by extracellular protons. 1 Homomeric ASIC1a channels are expressed throughout central and peripheral nervous systems and are implicated in learning/memory, pain sensation and neuronal death. 1-8 Among all these functions, mediating ischemic neuronal death was one of the most prominent pathological features of ASIC1a channels. [5][6][7] Although it has been established that severe extracellular acidosis in ischemic brain overactivated ASIC1a and caused neuronal death, 5,6 the death mechanisms remained largely unknown particularly considering that ASIC1a channels completely desensitize within a few seconds during persistent acidosis. 9 Thus, under such ischemic conditions, there might be other ASIC1a-associated mechanism(s) than acidosisinduced channel activation to explain the prominent effect against ischemic insult in the ASIC1a gene deletion mutant. 5 Indeed, ischemic neuronal death is a consequence of numerous ionic, biochemical and cellular events. 10,11 Multiple causes have been identified for neuronal demise such as excitotoxicity, acidotoxicity, oxidative stress and inflammation. [10][11][12] The ASIC1a channels may be involved in one or more of these mechanisms.Mitochondria are the key death executors in the cell, playing a central role in neuronal damages associated with neurological disorders such as ischemic stroke and neurodegenerative diseases. 13 Mitochondrial permeability transition (MPT) is a key event that occurs in most forms of cell demise ...

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Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

Cited by 57 publications

References 36 publications

Insights into the Mechanism of Pore Opening of Acid-sensing Ion Channel 1A

Insights into the Mechanism of Pore Opening of Acid-sensing Ion Channel 1A

Proton-mediated Conformational Changes in an Acid-sensing Ion Channel

Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death

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