BPS2026 – Structural and functional characterization of Vibrio cholerae mechanosensitive channel MSCs

Seasonal cholera outbreaks in areas lacking potable, clean water are common, largely due to the high stability of Vibrio cholerae in freshwater. V. cholerae , a typical inhabitant of shellfish chitin surfaces in brackish water, can easily adapt to both high and low salinity environments. This efficient adaptation to low-osmolarity streams or rainwater occurs because of a robust set of mechanosensitive channels (MSCs) in the genome, including one MscL-type channel and other MSCs family members that include one close homolog of E. coli MSCs. During a sudden osmotic decrease, these channels open sequentially, releasing small metabolites that reduce the osmotic gradient and prevent cell lysis. Unlike MscL, which act as a simple two-state emergency release valve, V. cholerae MSCs ( Vc MSCs) exhibits complex adaptive three-state behavior which includes inactivation that prevents excessive metabolite loss and terminates the immense hypoosmotic permeability increase by reducing the tension below the MscL threshold. Vc MSCs has been identified and studied using bacterial patch-clamp electrophysiology; however, its structure has not been determined previously. In this study, we cloned, expressed, purified, and examined the Vc MSCs channel using electron microscopy. We use traditional detergent extraction in parallel with polymer extraction to preserve the native lipids associated with the MSCs protein complex. Comparing the native lipid environment to the non-native detergent provides insights into how lipids influence the adaptive gating mechanism of the MSCs channel, characterized by rapid opening and delayed inactivation transitions.