Investigating the stoichiometry and gating of DEG/ENaC channels with structural-functional studies

• Applicant: Dr. Sylvia Fechner
• Subject Area: Biophysics
• Term: from 2016 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 313913559

During sensory transduction, stimuli are detected, processed and transmitted to the nervous system. Ion channels convert physical stimuli into electrical signals and are the leading actors in sensory transduction. During touch, mechanoelectrical transduction (MeT) channels transform mechanical stimuli into an electrical responses and are well characterized in the touch receptor neurons (TRNs) of the nematode Caenorhabditis elegans. The proteins forming the MeT channels in TRNs belong to the conserved superfamily of DEG/ENaC/ASIC proteins that form trimeric channel complexes in neurons and other tissues. Two DEG/ENaC proteins present in the TRN MeT channel are known; a third candidate subunit was recently discovered, but its contribution to in vivo function is unclear. While their in vivo composition is not known, even less is known about how these channels harness the energy carried by touch to catalyze channel activation or gating.To investigate the gating behavior of the MeT channel complex, I will use the following experimental strategy: 1. Identify all channel pore-forming subunits: a) Investigate the effect of the loss of the new candidate subunit on touch sensation and by engineering mutations in the pore region with CRISPR/Cas9. I will characterize the effect of these mutations on TRN mechanotransduction in vivo with the FALCON setup, which combines in vivo electrophysiology with force feedback-controlled mechanical stimulation. b) Investigate MeT channel stoichiometry with imaging techniques using fluorescence tagged subunits and fluorescence complementation, in which two candidate subunits are tagged with one half of a fluorophore. Here, an increase in fluorescence would indicate that the tagged proteins are in close proximity. 2. Determine how certain channel domains contribute to mechanical activation in vivo: I will use the FALCON setup to investigate the change in MeT currents upon channel mutations in the second transmembrane domain, which is known to be crucial for channel function.The gained knowledge will enhance the understanding not only of sensory mechanotransduction in C. elegans, but also of other members of the DEG/ENaC/ASIC channel family and beyond that our fundamental understanding of how all mechanosensitive ion channels respond to touch in vivo.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Dr. Miriam Beth Goodman, Ph.D.