Highly Conserved Salt Bridge Stabilizes Rigid Signal Patch at Extracellular Loop Critical for Surface Expression of Acid-sensing Ion Channels

Yang, Yang; Yu, Ye; Jin, Cheng; Liu, Yan; Liu, Di Shi; Wang, Jin; Zhu, Michael X.; Wang, Rui; Xu, Tian

doi:10.1074/jbc.m111.334250

Cited by 17 publications

(16 citation statements)

References 51 publications

(60 reference statements)

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“…Cell Surface Biotinylation and Western Blot Analysis-Surface biotinylation and Western blots were performed on cultured HEK293 cells according to our previous protocols (26,27). The HEK 293 cells were transfected with 8 g plasmids of HaFaNaC or its mutants (in pRc-CMV vector), 1.2 g plasmid of green fluorescent protein (as a transfection marker), and 15 l transfection reagent Hylimax.…”

Section: Methodsmentioning

confidence: 99%

“…First introduced into protein engineering in 1984 (32), double mutant cycle analysis was used to examine intramolecular and intermolecular interactions between proteins or a protein and its ligands (27,33,34). A double-mutant cycle involves a WT protein, two single mutants, and the corresponding double mutant protein.…”

Section: Double Mutant Cycle Analysis Indicates a Cooperative Mechanimentioning

confidence: 99%

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Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

Niu

Yang

Liu

et al. 2016

Journal of Biological Chemistry

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FMRFamide (Phe-Met-Arg-Phe-NH 2 )-activated sodium channel (FaNaC) is an amiloride-sensitive sodium channel activated by endogenous tetrapeptide in invertebrates, and belongs to the epithelial sodium channel/degenerin (ENaC/DEG) superfamily. The ENaC/DEG superfamily differs markedly in its means of activation, such as spontaneously opening or gating by mechanical stimuli or tissue acidosis. Recently, it has been observed that a number of ENaC/DEG channels can be activated by small molecules or peptides, indicating that the ligand-gating may be an important feature of this superfamily. The peptide ligand control of the channel gating might be an ancient ligand-gating feature in this superfamily. Therefore, studying the peptide recognition of FaNaC channels would advance our understanding of the ligand-gating properties of this superfamily of ion channels. Here we demonstrate that Tyr-131, Asn-134, Asp-154, and Ile-160, located in the putative upper finger domain of Helix aspersa FaNaC (HaFaNaC) channels, are key residues for peptide recognition of this ion channel. Two HaFaNaC specificinsertion motifs among the ENaC/DEG superfamily, residing at the putative ␣4-␣5 linker of the upper thumb domain and the ␣6-␣7 linker of the upper knuckle domain, are also essential for the peptide recognition of HaFaNaC channels. Chemical modifications and double mutant cycle analysis further indicated that those two specific inserts and key residues in the upper finger domain together participate in peptide recognition of HaFaNaC channels. This ligand recognition site is distinct from that of acid-sensing ion channels (ASICs) by a longer distance between the recognition site and the channel gate, carrying useful information about the ligand gating and the evolution of the trimeric ENaC/DEG superfamily of ion channels.The epithelial sodium channel/Degenerin (ENaC/DEG) 4 superfamily, cloned in the early 1990s, is nonvoltage-gated, amiloride-sensitive, and sodium-selective ion channels, including ASIC, ENaC, DEG, FaNaC, bile acid-sensitive ion channel (BASIC), and pickpocket (PPK) channels, etc. (1, 2). ENaC/DEG ion channels are implicated in many physiological and pathological functions such as synaptic plasticity, learning and memory, emotion regulation, neurodegenerative diseases, epileptic seizures, pain sensation, mechano-sensation, blood pressure regulation, and cystic fibrosis (1-3), which makes them potential drug targets for those disorders. The members of the ENaC/ DEG superfamily differ markedly by means of activation. For example, DEG channels are mechano-sensitive; ENaC channels open spontaneously; ASIC channels are capable of sensing tissue acidosis, while FaNaC is activated by RFamide peptides, etc. (4). Recently, great advances have been made in the exploration of the activation mechanism of this superfamily. For example, it is now known that ENaC, ASIC3, BASIC, and ASIC1a channels can be activated by the small molecules S3969 (5), GMQ (6), bile acid (7), and peptide toxin (8, 9) respectively. These new findings sugge...

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Section: Methodsmentioning

confidence: 99%

Section: Double Mutant Cycle Analysis Indicates a Cooperative Mechanimentioning

confidence: 99%

Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

Niu

Yang

Liu

et al. 2016

Journal of Biological Chemistry

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“…Cell surface biotinylation and western blotting were performed as we previously described 24 . In brief, the HEK-293 cells transfected with rP2X4 or its mutants were washed in chilled PBS þ / þ , and then incubated with sulfo-NHS-LC-biotin.…”

Section: Methodsmentioning

confidence: 99%

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

Zhao¹,

Wang

Ma³

et al. 2014

Nat Commun

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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.

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“…This interaction is the most commonly observed interaction to contribute to the stability of the conformation of proteins (46 -48). Previous studies have demonstrated that a salt bridge is implicated in many protein functions, such as the ligand binding of the GABA A receptor (33), the stabilization of the activation state of Kir1.1 (37), the folding of the serotonin transporter (38), the surface expression of ASIC channels (34), and the protein thermostability of enzymes (36). Here, we suggest that the salt bridge formed between Arg-309 and Asp-85 played an essential role in the protein stability and channel gating of P2X4 receptors.…”

Section: Discussionmentioning

confidence: 99%

“…Salt bridges, commonly formed by a combination of noncovalent interactions (hydrogen bonding and electrostatic interactions) between acidic and alkaline amino acids, play important roles in maintaining the structures and functions of enzymes, receptors, and other proteins (30 -38). Previous studies have demonstrated important roles of salt bridges in channel functions, such as channel gating (31) and protein expression (34), in a wide variety of ion channels. Therefore, the reason why the polymorphism of one residue of this salt bridge induced loss-of-function in human P2X receptors should be reevaluated.…”

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confidence: 99%

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

Zhao

Sun

et al. 2016

Journal of Biological Chemistry

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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 ␤2,3-sheet that is structurally coupled with this salt bridge and the ␣2-helix.

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Highly Conserved Salt Bridge Stabilizes Rigid Signal Patch at Extracellular Loop Critical for Surface Expression of Acid-sensing Ion Channels

Cited by 17 publications

References 51 publications

Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

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

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

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