Open structure and gating of the Arabidopsis mechanosensitive ion channel MSL10

Abstract

Plants are challenged by drastically different osmotic environments during growth and development. Adaptation to these environments often involves mechanosensitive ion channels that can detect and respond to mechanical force. In the model plant Arabidopsis thaliana, the mechanosensitive channel MSL10 plays a crucial role in hypo-osmotic shock adaptation and programmed cell death induction, but the molecular basis of channel function remains poorly understood. Here, we report a structural and electrophysiological analysis of MSL10. The cryo-electron microscopy structures reveal a distinct heptameric channel assembly. Structures of the wild-type channel in detergent and lipid environments, and in the absence of membrane tension, capture an open conformation. Furthermore, structural analysis of a non-conductive mutant channel demonstrates that reorientation of phenylalanine side chains alone, without main chain rearrangements, may generate the hydrophobic gate. Together, these results reveal a distinct gating mechanism and advance our understanding of mechanotransduction.

Fig. 1. Cryo-EM structure of AtMSL10.

a Cryo-EM reconstruction of the wild-type full-length channel in detergents, with each subunit uniquely colored. The unsharpened map contoured at a lower level (in black) illustrates the detergent micelle densities surrounding the transmembrane domain (TMD). Notably, densities for the N-terminal ‘death’ domain (residues 1–165) are not resolved. b The heptameric channel architecture. c A single channel subunit, with TM helices 1–6, cytoplasmic linker domain (CLD), and C-terminal cytoplasmic domain (CTD) highlighted.

Fig. 2. Structural comparison of MscS homologs.

a Protomer structures of AtMSL10, the ‘down’ and ‘up’ conformations of DmMSL10/FLYC1 (PDB: 7N5D), EcMscS (PDB: 6RLD), and AtMSL1 (PDB: 6VXM). TMD, CTD, and CLD are labeled. b Closeup views of the boxed regions from (a). The corresponding residues forming a salt bridge in the ‘up’ conformation of DmFLYC1 are shown as sticks. The side chain of N316 in AtMSL10, which corresponds to R334 in DmFLYC1, was not resolved in the cryo-EM density map. The cytoplasmic linker domain (CLD) between TM4 and TM5 in AtMSL10 resembles the ‘down’ conformation of CLD in DmFLYC1.

Fig. 3. Open conformation of the wild-type AtMSL10.

a The ion conduction pore of AtMSL10. Only two opposing subunits are shown for clarity, and blue dots outline the central pore. The narrow positions at V549 and F553 are highlighted. b Pore dimension. The pore profile of the wild-type AtMSL10 (dark blue) is compared with those of DmMSL10/FLYC1 (light blue, PDB:7N5D) and open (red, PDB:2VV5) and closed (green, PDB: 6RLD) EcMscS. c The ion pore of DmMSL10/FLYC1, with the narrow constriction at F572 labeled. d Superposition of the structures of AtMSL10 in detergents (dark blue) and in saposin lipid nanoparticles (salmon).

Fig. 4. Domain organization of MscS homologs.

The structures of AtMSL10, DmMSL10/FLYC1 (PDB: 7N5D), EcMscS (PDB: 6RLD) and AtMSL1 (PDB: 6VXM) are shown. In each of the heptameric channels, a single subunit is uniquely colored to illustrate domain arrangements.

Fig. 5. Hydrophobic gate closure.

a Overlay of the structures of the wild-type AtMSL10 (blue) and G556Vmutant (orange). b Comparison of the ion pore profiles of the wild-type (blue) and G556V mutant (orange) channels. c Superposition of the pore lining helices of the wild type (blue) and G556V (orange). Amino acids 553 and 556 are highlighted as sticks. The side chain reorientation of F553 is indicated by a red arrow.