A selectivity filter at the intracellular end of the acid-sensing ion channel pore
Abstract
Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the 'GAS belt' in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.
Article and author information
Author details
Funding
Lundbeckfonden (Lundbeck Foundation Fellowship R139-2012-12390)
- Stephan A Pless
Carlsbergfondet (Equipment Grant 2013_01_0439)
- Stephan A Pless
Det Frie Forskningsråd (Postdoctoral Fellowship 4092-00348B)
- Timothy Lynagh
Australian Research Council (Project Grant DP170101732)
- Toby W Allen
Novo Nordisk Foundation (Project Grant)
- Stephan A Pless
National Health and Medical Research Council (Project Grant APP1104259)
- Toby W Allen
National Institutes of Health (Project Grant U01-11567710)
- Toby W Allen
Lundbeckfonden (Postdoctoral Fellowship R171-2014-558)
- Timothy Lynagh
National Cancer Institute (dd7)
- Toby W Allen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in accordance with the recommendations by the by the Danish Veterinary and Food Administration and approved under license 2014−15−0201−00031. Surgery was performed on Xenopus laevis frogs anaesthetized in 0.3% tricaine.
Copyright
© 2017, Lynagh et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,672
- views
-
- 607
- downloads
-
- 55
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Structural Biology and Molecular Biophysics
In addition to its role as cellular energy currency, adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of membrane proteins that form heptameric large-pore channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Secretomics experiments reveal that lysophospholipid-activated pannexin 1 leads to the release of not only ATP but also other signaling metabolites, such as 5’-methylthioadenosine, which is important for immunomodulation. We also demonstrate that lysophospholipids activate endogenous pannexin 1 in human monocytes, leading to the release of IL-1β through inflammasome activation. Our results provide a connection between lipid metabolism and purinergic signaling, both of which play major roles in immune responses.
-
- Structural Biology and Molecular Biophysics
Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.