Structure of mechanically activated ion channel OSCA2.3 reveals mobile elements in the transmembrane domain

Figures

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  Figure 1.. Structure of OSCA2.3 and OSCA1.2 in peptidiscs. DeepEMhancer maps (a, d), side (b, e) and top (c, f) views of models of OSCA2.3 and OSCA1.2, respectively. Ex: Extracellular, In: Intracellular. g, Close-up view from c displaying the EICs. h, Right: Close-up view of the inter-subunit cleft of OSCA2.3 (salmon) and OSCA1.2 (blue). Black lines represent the approximate height at which cross-sectional area was measured. Left: Representation of the shape of the cross sections and measurements of the area occupied. i, Superposition of gaussian-filtered unsharpened maps showing difference in overall peptidisc shape of OSCA2.3 (red) and OSCA1.2 (blue).

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  Figure 2.. Pores and membrane fenestration. a, Superposition of pore lining TMs of OSCA1.2 and OSCA2.3 (left), with the movements of TM6a-TM7 cytoplasmic linker, TM0 and extracellular end of TM3 denoted by black arrows. Superposition of TM3 only (right), with kinked residues of OSCA2.3 colored light salmon. b, Superposition of pore lining TMs of TMEM16F F518H mutant structures with (PDB:8B8J) and without Ca2+ bound. (PDB:8B8J) (left), with the movements of the extracellular ends of TM3 and TM4 denoted by black arrows. Superposition of TM3 only of TMEM16F F518H mutants and OSCA2.3 (right). Depiction of pore pathway along the TMD of OSCA1.2 (c), OSCA2.3 (d) in peptidiscs and TMEM16F F518H Ca2+ bound (e). TM4 was omitted for clarity and the conserved glutamate residue of OSCAs and the π-helix tryptophan residue are shown as sticks and labeled. In green: pore lipid; light green: TM6a-TM7 linker and asterisks (*) denote π-helical turns. f, Pore radii profile of OSCA1.2 and OSCA2.3 in peptidiscs (PD), and TMEM16F F518H Ca2+ bound (PDB: 8B8J). Constriction caused by TM3 and secondary constriction is highlighted in light salmon and light green, respectively. g, Superposition of OSCA1.2 and OSCA2.3…

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  Figure 3.. Interactions of the TM6a-TM7 linker with the BLD and TM0. a, Side view of OSCA1.2 centered on interactions between the BLD, TM6b/IL4H and cytoplasmic helix after TM0. b, Contact map from coevolutionary sequence analysis. Each points represents a pair of position predicted to be evolutionary coupled, colored based on probability of the coupling. Pairings closer than 6 positions in the sequence or have a probability of less than or equal to 0.7 are omitted. Tan and ash grey colored regions represent intra and inter subunit contacts, respectively, in experimental structures that are, on average, 5 Å or less apart. Circled are the coupled pairs between the BLD and IL4H. Bar on top represents the position of each TM in the sequence. c, Evolutionary coupled residues between BLD and IL4H depicted in OSCA1.2 structure. Sequence logo (d) and alignment (e) for the BLD (top) and IL4H (bottom) region that contains the coupled residues (circled in b). Dashed lines in d and arrows in e denote the coupled residues, color coded as in c. f, Interface between intracellular helix of TM0 and TM6b/ILH4. Dashed lines represent apparent hydrogen bonds. g, Conservation of residues in f between OSCA1.2…

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  Figure 4.. Proposed states of OSCA channels. In the absence of mechanical stimulus, the channel is in a resting state, with the hydrophobic gate closed and lipids populating the membrane fenestration. Upon membrane tension, the BLD or amphipathic helix (IL1H1) transmit the stimulus to the TMD, accompanied by delipidation of the pore pathway and the channel reorganizes into an unknown open state. Continuous stimulus causes the channel to inactivate, presumably by action of the TM6b-TM7 linker. Presumably, after the mechanical stimulus ceases, the hydrophobic gate closes, reaching the intermediate state represented by the OSCA2.3 structure in this study. Finally, the channel enters a resting state after lipids repopulate the pore fenestration.