Investigating the stoichiometry and gating of DEG/ENaC channels with structural-functional studies
Final Report Abstract
Touch receptor neurons (TRNs) of C. elegans are used as a system to understand the molecular events responsible for touch sensation. Key actors in this process include DEG/ENaC/ASIC proteins. These proteins form a mechanoelectrical transduction (MeT) channel: MEC-4 is an essential, pore-forming subunit, MEC-10 is a non-essential, regulatory subunit. These two proteins are co-expressed with DEGT-1, another homologous protein. The presence of a third protein re-opens the question how those three proteins co-assemble to form the MeT channels in vivo. We are using a combination of behavioral assays, in vivo patch-clamp recording, and heterologous expression to investigate the contribution of DEGT-1 to touch and MeT channel formation. Classical touch assays revealed that degt-1;mec-10 double mutants have a more severe phenotype than degt-1 and mec-10 single mutants. With in vivo patch-clamp recordings of native MeT currents activated by feedback-controlled mechanical stimulation, we identified a similar genetic enhancement: the maximal amplitude of the MeT currents was further reduced in degt-1;mec-10 double mutants than single mutants. Thus, DEGT-1 most likely plays a role in the formation of the native MeT-channel complex. To characterize DEGT-1-containing channels independent of the native tissue, we introduced the well-known d mutation and expressed DEGT-1d alone and in combination with MEC-4d in Xenopus oocytes. We found that DEGT-1d forms homomeric channels whose properties differ from those of MEC-4d. As found for MEC-10d, co-expressing DEGT-1d with MEC-4d decreased current amplitude compared to MEC-4d alone. Collectively, these results support the conclusion that DEGT-1, like MEC-10, plays a regulatory role in MeT channel formation and function.
Publications
- Microfluidics for Studying Mechanobiology in Model Organisms, Methods in Cell Biology Volume 146, 2018, Pages 217-259
AA. Kim, AL Nekimken, S. Fechner, LE O’Brien and BL Pruitt
(See online at https://doi.org/10.1016/bs.mcb.2018.05.010)
