Structure of an acid-sensing ion channel in a lipid environment.
EMSL Project ID
50929
Abstract
Acid-sensing ion channels (ASICs) are voltage-insensitive and proton-gated members of the Na-selective epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout central and peripheral nervous systems. The homotrimeric ASIC1a channel is particularly relevant to central nervous system processes, has been implicated in both nociception and fear conditioning, and as such, is a viable target for the development of novel therapeutics. While ASIC1a channels are the most structurally well-characterized of the ENaC/DEG family of ion channels and both X-ray and cryo-EM structures of detergent solubilized chicken ASIC1a channels in the three primary functional states: high pH resting1, low pH open2 and low pH desensitized3,4 have been solved, the architecture of the channel’s cytoplasmic terminal domains remains unknown. The ultimate goal of this research is to elucidate the architecture of a full-length ASIC in a membrane-like environment, thus preserving the integrity of the transmembrane domain and revealing the architecture of the channel’s cytosolic termini. Notably, the amino terminus of ASICs in particular is highly conserved across the entire ENaC/DEG superfamily of ion channels and harbors sites for disease causing mutations as well as motifs critical for channel gating. In particular, a conserved ‘His-Gly’ motif has been shown to be indispensable for gating in ENaCs and when mutated, can disrupt the ability to regulate sodium levels in the body5. Despite the numerous high-resolution structures of ASIC1a solubilized in detergent micelles, however, no density for the amino terminus or the HG motif has been observed. Therefore, in addition to high-resolution reconstructions of chicken ASIC1a channels in a membrane-like environment, the results from this proposal will yield valuable structural insights relevant to the entire ENaC/DEG superfamily of ion channels. To accomplish the goals outlined in this proposal, we are requesting time on a Titan Krios transmission electron microscope to image vitrified samples of chicken ASIC1a channels maintained in a lipid environment and in multiple pH-dependent functional states for single-particle cryo-electron microscopy studies. Given our promising preliminary results, we are confident that these resources will be sufficient to yield high-resolution reconstructions and will generate important structural insights into the terminal domain architecture and the relevance of the elusive ‘HG’ motif to proton-dependent gating. 1 Yoder, N., Yoshioka, C. & Gouaux, E. Gating mechanisms of acid-sensing ion channels. Nature 555, 397-401, (2018). 2 Baconguis, I., Bohlen, C. J., Goehring, A., Julius, D. & Gouaux, E. X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na(+)-selective channel. Cell 156, 717-729, (2014). 3 Jasti, J., Furukawa, H., Gonzales, E. B. & Gouaux, E. Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH. Nature 449, 316-323, (2007). 4 Gonzales, E. B., Kawate, T. & Gouaux, E. Pore architecture and ion sites in acid-sensing ion channels and P2X receptors. Nature 460, 599-604, (2009). 5 Grunder, S. et al. A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel. EMBO J 16, 899-907, (1997).
Project Details
Start Date
2019-06-15
End Date
2019-12-14
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members