Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft.
The epithelial Na(+) channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na(+), Cl(-), protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na(+) concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na(+) binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na(+). Mutations at selected sites altered the cation inhibitory preference to favor Li(+) or K(+) rather than Na(+). Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na(+). Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.
From the Departments of Medicine and obk2@pitt.edu.
Ossama B
OB
Kashlan
From the Departments of Medicine and.
Brandon M
BM
Blobner
From the Departments of Medicine and.
Zachary
Z
Zuzek
From the Departments of Medicine and.
Michael
M
Tolino
From the Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Thomas R
TR
Kleyman
D062565:Acid Sensing Ion Channels / Q000737:chemistry* / Q000235:genetics; D000200:Action Potentials; D000494:Allosteric Regulation; D000584:Amiloride / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D062686:Epithelial Sodium Channel Blockers / Q000737:chemistry; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D017136:Ion Transport; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D012964:Sodium / Q000737:chemistry*; D014982:Xenopus laevis
C568267:ASIC1 protein, mouse; D062565:Acid Sensing Ion Channels; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D000584:Amiloride; D012964:Sodium
Acid-sensing Ion Channel (ASIC); Allosteric Regulation; Effector Specificity; Epithelial Sodium Channel (ENaC); Hypertension; Ion Channel; Membrane Transport; Protein Cross-linking; pH-dependent Activation