Characterization and Physiological Role of a Bacterial-Like Mechanosensitive Channel in Trypanosoma Cruzi Osmoregulation

Abstract

that the C-terminus is involved in channel gating. The mutation E2117D slowed the inactivation rate 8-fold. We made heteromeric channels in HEK cells by co-transfecting with high and low conductance mutants. This created active channels with two new unitary conductances indicating that pore formation occurs at the subunits' interface. The number of new conducting states supports the trimer structure as revealed by Cryo EM. This is the first demonstration of functional heteromers of Piezo channels. Heteromeric formation is likely to be important for understanding the physiological activity of these channels. 473-Pos Board B253Pore Determinants of Mechanosensitive Piezo Channels Piezo proteins have been proposed as the long-sought-after mechanosensitive (MS) cation channels in mammals that play critical roles in various mechanotransduction processes, such as touch sensation and vascular development. However, their ion-conducting pore and ion permeation mechanisms have remained undefined. Here we identify domains and specific residues in Piezo1 that control the essential pore properties, including unitary conductance, ion selectivity and pore blockage, pinpointing the ion-conducting pathway. By uncovering the bona fide ion-conducting pore, these findings not only provide definitive proof that Piezo proteins are genuine pore-forming subunits of MS cation channels, but also shed light on elucidating the ion permeation and gating mechanisms of this prototypic class of mammalian MS cation channels. 474-Pos Board B254In order to complete its life cycle, Trypanosoma cruzi-the protozoan parasite that causes Chagas disease-faces various environmental changes as it propagates from an insect vector to a mammalian host. Previous studies have shown that T. cruzi has a robust osmoregulatory response, however the osmosensors involved in the detection and compensation pathways have not been identified. Mechanosensitive channels, which are activated by a stretch of the plasma membrane, have been associated with sensing of environmental changes in other organisms, but the function of these channels in T. cruzi is still unknown. In silico analysis of T. cruzi genome reveals the presence of mechanosensitive channels similar to the ones described in bacteria. We hypothesize that a bacterial-like mechanosensitive channel, TcMcS, is involved in osmoregulatory processes in T. cruzi. Overexpressing mutants using a tetracycline inducible system were developed to investigate the role of TcMcS in T. cruzi osmoregulation. Knockout mutants mediated by CRISPR/Cas9 were generated to test the essentiality of the protein.The localization and expression pattern of TcMcS varied in the three main life stages of T. cruzi. TcMcS seems to be localized in the contractile vacuole of epimastigote and trypomastigote forms, and in the plasma membrane of intracellular amastigote forms. Under hyposmotic stress, cells overexpressing TcMcS swell significantly less than wild-type parasites. Under EGTA treatment, this advantage was eliminated, suggesting that calci...