Ye Yu

Extracellular Spermine Exacerbates Ischemic Neuronal Injury through Sensitization of ASIC1a Channels to Extracellular Acidosis

Ischemic brain injury is a major problem associated with stroke. It has been increasingly recognized that acid-sensing ion channels (ASICs) contribute significantly to ischemic neuronal damage, but the underlying mechanism has remained elusive. Here, we show that extracellular spermine, one of the endogenous polyamines, exacerbates ischemic neuronal injury through sensitization of ASIC1a channels to extracellular acidosis. Pharmacological blockade of ASIC1a or deletion of the ASIC1 gene greatly reduces the enhancing effect of spermine in ischemic neuronal damage both in cultures of dissociated neurons and in a mouse model of focal ischemia. Mechanistically, spermine profoundly reduces desensitization of ASIC1a by slowing down desensitization in the open state, shifting steady-state desensitization to more acidic pH, and accelerating recovery between repeated periods of acid stimulation. Spermine-mediated potentiation of ASIC1a activity is occluded by PcTX1 (psalmotoxin 1), a specific ASIC1a inhibitor binding to its extracellular domain. Functionally, the enhanced channel activity is accompanied by increased acid-induced neuronal membrane depolarization and cytoplasmic Ca2+ overload, which may partially explain the exacerbated neuronal damage caused by spermine. More importantly, blocking endogenous spermine synthesis significantly attenuates ischemic brain injury mediated by ASIC1a but not that by NMDA receptors. Thus, extracellular spermine contributes significantly to ischemic neuronal injury through enhancing ASIC1a activity. Our data suggest new neuroprotective strategies for stroke patients via inhibition of polyamine synthesis and subsequent spermine–ASIC interaction.

Serotonin Facilitates Peripheral Pain Sensitivity in a Manner That Depends on the Nonproton Ligand Sensing Domain of ASIC3 Channel

Tissue acidosis and inflammatory mediators play critical roles in inflammatory pain. Extracellular acidosis activates acid-sensing ion channels (ASICs), which have emerged as key sensors for extracellular protons in the central and peripheral nervous systems and play key roles in pain sensation and transmission. Additionally, inflammatory mediators, such as serotonin (5-HT), are known to enhance pain sensation. However, functional interactions among protons, inflammatory mediators, and ASICs in pain sensation are poorly understood. In the present study, we show that 5-HT, a classical pro-inflammatory mediator, specifically enhances the proton-evoked sustained, but not transient, currents mediated by homomeric ASIC3 channels and heteromeric ASIC3/1a and ASIC3/1b channels. Unexpectedly, the effect of 5-HT on ASIC3 channels does not involve activation of 5-HT receptors, but is mediated via a functional interaction between 5-HT and ASIC3 channels. We further show that the effect of 5-HT on ASIC3 channels depends on the newly identified nonproton ligand sensing domain. Finally, coapplication of 5-HT and acid significantly increased pain-related behaviors as assayed by the paw-licking test in mice, which was largely attenuated in ASIC3 knock-out mice, and inhibited by the nonselective ASIC inhibitor amiloride. Together, these data identify ASIC3 channels as an unexpected molecular target for acute actions of 5-HT in inflammatory pain sensation and reveal an important role of ASIC3 channels in regulating inflammatory pain via coincident detection of extracellular protons and inflammatory mediators.

Atomic level characterization of the nonproton ligand-sensing domain of ASIC3 channels.

Acid-sensing ion channels (ASICs) are known to be primarily activated by extracellular protons. Recently, we characterized a novel nonproton ligand (2-guanidine-4-methylquinazoline, GMQ), which activates the ASIC3 channel subtype at neutral pH. Using an interactive computational-experimental approach, here we extend our investigation to delineate the architecture of the GMQ-sensing domain in the ASIC3 channels. We first established a GMQ binding mode and revealed that residues Glu-423, Glu-79, Leu-77, Arg-376, Gln-271, and Gln-269 play key roles in forming the GMQ-sensing domain. We then verified the GMQ binding mode using ab initio calculation and mutagenesis and demonstrated the critical role of the above GMQ-binding residues in the interplay among GMQ, proton, and Ca2+ in regulating the function of ASIC3. Additionally, we showed that the same residues involved in coordinating GMQ responses are also critical for activation of the ASIC3E79C mutant by thiol-reactive compound DTNB. Thus, a range of complementary techniques provide independent evidence for the structural details of the GMQ-sensing domain at atomic level, laying the foundation for further investigations of endogenous nonproton ligands and gating mechanisms of the ASIC3 channels.

Nonproton Ligand Sensing Domain Is Required for Paradoxical Stimulation of Acid-sensing Ion Channel 3 (ASIC3) Channels by Amiloride

Background: Acid-sensing ion channels (ASICs) are activated by extracellular protons and are inhibited by amiloride. Results: Amiloride activates and sensitizes ASIC3 channels depending on the nonproton ligand sensing domain. Conclusion: ASICs can sense nonproton ligands in addition to protons. Significance: The results indicate caution in the use of amiloride for studying ASIC physiology and in the development of amiloride-derived ASIC inhibitors for treating pain syndromes. Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of epithelial sodium channel/degenerin. In this study, we reported that AMI paradoxically opened homomeric ASIC3 and heteromeric ASIC3 plus ASIC1b channels at neutral pH and synergistically enhanced channel activation induced by mild acidosis (pH 7.2 to 6.8). The characteristic profile of AMI stimulation of ASIC3 channels was reminiscent of the channel activation by the newly identified nonproton ligand, 2-guanidine-4-methylquinazoline. Using site-directed mutagenesis, we showed that ASIC3 activation by AMI, but not its inhibitory effect, was dependent on the integrity of the nonproton ligand sensing domain in ASIC3 channels. Moreover, the structure-activity relationship study demonstrated the differential requirement of the 5-amino group in AMI for the stimulation or inhibition effect, strengthening the different interactions within ASIC3 channels that confer the paradoxical actions of AMI. Furthermore, using covalent modification analyses, we provided strong evidence supporting the nonproton ligand sensing domain is required for the stimulation of ASIC3 channels by AMI. Finally, we showed that AMI causes pain-related behaviors in an ASIC3-dependent manner. These data reinforce the idea that ASICs can sense nonproton ligands in addition to protons. The results also indicate caution in the use of AMI for studying ASIC physiology and in the development of AMI-derived ASIC inhibitors for treating pain syndromes.

Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation

Channel gating in response to extracellular ATP is a fundamental process for the physiological functions of P2X receptors. Here we identify coordinated allosteric changes in the left flipper (LF) and dorsal fin (DF) domains that couple ATP-binding to channel gating. Engineered disulphide crosslinking or zinc bridges between the LF and DF domains that constrain their relative motions significantly influence channel gating of P2X4 receptors, confirming the essential role of these allosteric changes. ATP-binding-induced alterations in interdomain hydrophobic interactions among I208, L217, V291 and the aliphatic chain of K193 correlate well with these coordinated relative movements. Mutations on those four residues lead to impaired or fully abolished channel activations of P2X4 receptors. Our data reveal that ATP-binding-induced altered interdomain hydrophobic interactions and the concomitant coordinated motions of LF and DF domains are allosteric events essential for the channel gating of P2X4 receptors.

Role of Acid-Sensing Ion Channel 1a in the Secondary Damage of Traumatic Spinal Cord Injury

Objective: To determine the cellular and molecular mechanisms by which acid-sensing ion channel 1a (ASIC1a) plays its role in the secondary injury after traumatic spinal cord injury (SCI), and validate the neuroprotective effect of ASIC1a suppression in SCI model in vivo. Background: Secondary damage after traumatic SCI contributes to the exacerbation of cellular insult and thereby contributes to spinal cord dysfunction. However, the underlying mechanisms remain largely unknown. Acidosis is commonly involved in the secondary injury process after the injury of central nervous system, but whether ASIC1a is involved in secondary injury after SCI is unclear. Methods: Male Sprague–Dawley rats were subjected to spinal contusion using a weight-drop injury approach. Western blotting and immunofluorescence assays were used to observe the change of ASIC1a expression after SCI. The TUNEL staining in vivo as well as the cell viability and death assays in spinal neuronal culture were employed to assess the role of ASIC1a in the secondary spinal neuronal injury. The electrophysiological recording and Ca2+ imaging were performed to reveal the possible underlying mechanism. The antagonists and antisense oligonucleotide for ASIC1a, lesion volume assessment assay and behavior test were used to estimate the therapeutic effect of ASIC1a on SCI. Results: We show that ASIC1a expression is markedly increased in the peri-injury zone after traumatic SCI. Consistent with the change of ASIC1a expression in injured spinal neurons, both ASIC1a-mediated whole-cell currents and ASIC1a-mediated Ca2+ entry are significantly enhanced after injury. We also show that increased activity of ASIC1a contributes to SCI-induced neuronal death. Importantly, our results indicate that down-regulation of ASIC1a by antagonists or antisense oligonucleotide reduces tissue damage and promotes the recovery of neurological function after SCI. Conclusion: This study reveals a cellular and molecular mechanism by which ASIC1a is involved in the secondary damage process after traumatic SCI. Our results suggest that blockade of Ca2+-permeable ASIC1a may be a potential neuroprotection strategy for the treatment of SCI patients.

Inherent Dynamics of Head Domain Correlates with ATP-Recognition of P2X4 Receptors: Insights Gained from Molecular Simulations

P2X receptors are ATP-gated ion channels involved in many physiological functions, and determination of ATP-recognition (AR) of P2X receptors will promote the development of new therapeutic agents for pain, inflammation, bladder dysfunction and osteoporosis. Recent crystal structures of the zebrafish P2X4 (zfP2X4) receptor reveal a large ATP-binding pocket (ABP) located at the subunit interface of zfP2X4 receptors, which is occupied by a conspicuous cluster of basic residues to recognize triphosphate moiety of ATP. Using the engineered affinity labeling and molecular modeling, at least three sites (S1, S2 and S3) within ABP have been identified that are able to recognize the adenine ring of ATP, implying the existence of at least three distinct AR modes in ABP. The open crystal structure of zfP2X4 confirms one of three AR modes (named AR1), in which the adenine ring of ATP is buried into site S1 while the triphosphate moiety interacts with clustered basic residues. Why architecture of ABP favors AR1 not the other two AR modes still remains unexplored. Here, we examine the potential role of inherent dynamics of head domain, a domain involved in ABP formation, in AR determinant of P2X4 receptors. In silico docking and binding free energy calculation revealed comparable characters of three distinct AR modes. Inherent dynamics of head domain, especially the downward motion favors the preference of ABP for AR1 rather than AR2 and AR3. Along with the downward motion of head domain, the closing movement of loop139–146 and loop169–183, and structural rearrangements of K70, K72, R298 and R143 enabled ABP to discriminate AR1 from other AR modes. Our observations suggest the essential role of head domain dynamics in determining AR of P2X4 receptors, allowing evaluation of new strategies aimed at developing specific blockers/allosteric modulators by preventing the dynamics of head domain associated with both AR and channel activation of P2X4 receptors.

Highly conserved salt bridge stabilizes rigid signal patch at extracellular loop critical for surface expression of acid-sensing ion channels

Background: Plasma membrane expression is vital for the function of ASICs, which act as extracellular proton sensors. Results: Mutations in a conserved salt bridge and its adjacent region impaired cell surface ASIC expression. Conclusion: Surface ASIC expression involves an exposed rigid signal patch at the extracellular loop. Significance: This finding sheds lights on new strategies to prevent excessive neuronal excitability associated with ASIC activation. Acid-sensing ion channels (ASICs) are non-selective cation channels activated by extracellular acidosis associated with many physiological and pathological conditions. A detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In this study, we examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp107-Arg153) and human ASIC1a (Asp107-Arg160) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt bridge is essential for functional expression of ASICs in a pH sensing-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis, and further mutagenesis studies suggested a high stability and structural constrain of the salt bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation.

Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel

P2X receptors are non-selective cation channels gated by extracellular ATP, and the P2X7 receptor subtype plays a crucial role in the immune and nervous systems. Altered expression and dysfunctions of P2X7 receptors caused by genetic deletions, mutations, and polymorphic variations have been linked to various diseases, such as rheumatoid arthritis and hypertension. Despite the availability of crystal structures of P2X receptors, the mechanism of competitive antagonist action for P2X receptors remains controversial. Here, we determine the crystal structure of the chicken P2X7 receptor in complex with the competitive P2X antagonist, TNP-ATP. The structure reveals an expanded, incompletely activated conformation of the channel, and identified the unique recognition manner of TNP-ATP, which is distinct from that observed in the previously determined human P2X3 receptor structure. A structure-based computational analysis furnishes mechanistic insights into the TNP-ATP-dependent inhibition. Our work provides structural insights into the functional mechanism of the P2X competitive antagonist.P2X receptors are nonselective cation channels that are gated by extracellular ATP. Here the authors present the crystal structure of chicken P2X7 with its bound competitive antagonist TNP-ATP and give mechanistic insights into TNP-ATP dependent inhibition through further computational analysis and electrophysiology measurements.

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors.

Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt bridge in P2X receptors. Here, we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide cross-linking, we revealed the stringent requirement of this salt bridge in normal P2X4 channel function. This salt bridge may contribute to stabilizing the bending conformation of the β2,3-sheet that is structurally coupled with this salt bridge and the α2-helix. Strongly kinked β2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and loop β7,8 in P2X4 receptors. Disulfide cross-linking with directions opposing or along the bending angle of the β2,3-sheet toward the α2-helix led to loss-of-function and gain-of-function of P2X4 receptors, respectively. Further insertion of amino acids with bulky side chains into the linker between the β2,3-sheet or the conformational change of the α2-helix, interfering with the kinked conformation of β2,3, led to loss-of-function of P2X4 receptors. All these findings provided new insights in understanding the contribution of the salt bridge between Asp-85 and Arg-309 and its structurally coupled β2,3-sheet to the function of P2X receptors.