Richard Posert

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily-conserved family of ion channels whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila . The molecular features that tune homologous TMC ion channel complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here we present the single particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans . The complex is composed of two copies each of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex reveals differences in subunit composition and highlights conserved protein-lipid interactions, as well as other structural features, that together suggest a mechanism for TMC-mediated mechanosensory transduction. Significance Statement: One mechanism by which organisms sense their environment is through the perception of mechanical stimuli such as sound, touch, and vibration. Transmembrane channel-like (TMC) proteins are ion channels whose function has been linked to a variety of mechanosensitive processes, including hearing and balance in vertebrates and touch sensation in worms. The molecular mechanisms by which TMCs respond to mechanical stimuli are unknown. Here we present the structure of the TMC-2 complex isolated from worms. Comparison of the TMC-2 complex to the recently solved structure of the worm TMC-1 complex highlights common structural features that are likely important for sensing mechanical stimuli yet also illuminates key differences that may explain the distinct functional roles of TMC-1 and TMC-2 in the worm.

Chromatography is an essential family of assays for molecular biology and chemistry. Typically, only a qualitative assessment of peak height, position, and shape are sufficient to proceed. However, there are more advanced forms of chromatography that require integration of peak width, baseline subtraction, and precise peak position calling. To handle these precise workflows, instrument manufacturer software is complicated and arcane; this makes simpler day-to-day analyses inefficient. Additionally, each manufacturer reports the data in their own proprietary format, requiring that analysis is performed at instrument computers and making data sharing between labs difficult. Here we present Appia, a free, open-source chromatography processing and visualization package focused on making analysis, collaboration, and publication quick and easy.

The molecular bases of heteromeric assembly and link between Na⁺ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits – α, β, and γ – assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the α subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the α2 helix dividing two distinct regulatory sites: Na⁺ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na⁺ site via the α2 helix highlighting the critical role of the α2 helix in regulating ENaC function.

The epithelial sodium channel (ENaC), a member of the ENaC/DEG superfamily, regulates Na⁺ and water homeostasis. ENaCs assemble as heterotrimeric channels that harbor protease-sensitive domains critical for gating the channel. Here, we present the structure of human ENaC in the uncleaved state determined by single-particle cryo-electron microscopy. The ion channel is composed of a large extracellular domain and a narrow transmembrane domain. The structure reveals that ENaC assembles with a 1:1:1 stoichiometry of α:β:γ subunits arranged in a counter-clockwise manner. The shape of each subunit is reminiscent of a hand with key gating domains of a ‘finger’ and a ‘thumb.’ Wedged between these domains is the elusive protease-sensitive inhibitory domain poised to regulate conformational changes of the ‘finger’ and ‘thumb’; thus, the structure provides the first view of the architecture of inhibition of ENaC.