Structural Biology and Molecular Biophysics

Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli

Sebastian Jojoa-Cruz
Adrienne E. Dubin
Wen-Hsin Lee
Andrew Ward author has email address

Department of Integrative Structural and Computational Biology, Scripps Research, La Jolla, California 92037, USA
Department of Neuroscience, Scripps Research, La Jolla, California 92037, USA

https://doi.org/10.7554/eLife.93147.2

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Figures and data

Cryo-EM structure and pore of OSCA3.1.
a, Cryo-EM map of OSCA3.1 dimer colored by subunit. Nanodisc density in grey corresponds to the unsharpened map (gaussian-filtered to 1.5 σ). b, Front (left) and top (right) view of atomic model. Ex.: Extracellular, In.: Intracellular. c, Superposition of OSCA1.1, OSCA1.2 (in nanodiscs) and OSCA3.1 protomers. d, Pore profile of OSCAs in c. e, View of the pore pathway (blue) of OSCA3.1. Pore facing residues colored in yellow, with selected residues labeled. π-helical turns in pink. Putative pore lipid in green. Backbone of TM3 and TM4 helices hidden for clarity.

Amphipathic helix mutants of OSCA1.2.
a, Superposition of OSCA1.2 (grey) and OSCA3.1 (red). Insets: close-up view of amphipathic helix. Residues substituted in OSCA1.2 for electrophysiology experiments are underlined. b, Few cells expressing mutations in the amphipathic helix respond to poking compared to WT controls. Top panel: Maximum poke-induced currents observed in whole-cell mode for cells exposed to displacements up to 8.9 ± 0.6 μm (N=51; mean ± S.E.M.), 12.8 ± 0.7 μm (N=24), 12.4 ± 0.7 μm (N=16), and 12.8 ± 0.5 μm (N=24) above touching for WT, OSCA1.2_W75K, OSCA1.2_L80E, and OSCA1.2_W75K/L80E, respectively. Bottom panel: the apparent threshold in μm above touching the cell for this cohort. c, SAC maximal current (I_max) (top) and mmHg threshold (bottom) from WT and OSCA1.2_W75K/L80E-expressing cells reveal no significant differences in the ability of negative pressure to activate channels in cell-attached patches (Student’s t-test). Also shown are data from single mutants OSCA1.2_W75K and OSCA1.2_L80E. Too few patches were obtained for OSCA1.2_W75K to compare I_max. Electrode resistances for c were similar in all cases (WT: 2.3 ± 0.1 MΩ (N=26); W75K: 2.1 ± 0.4 MΩ (N=3), L80E: 2.2 ± 0.1 MΩ (N=5), W75K/L80E: 1.8 ± 0.1 MΩ (N=11)). For panels b-c: individual cells are represented as scatter points; mean and S.E.M. are displayed.

OSCA1.2_OSCA3.1-BLD chimera.
a, Amino acid sequence alignment of the BLD region of OSCA1.2 and OSCA3.1. Full alignment in Supplementary Fig. 3. Blue line at bottom of sequences denotes the sequence swapped in OSCA1.2_OSCA3.1-BLD chimera. b, Poke-induced responses are observed in HEK-P1KO cells expressing OSCA1.2_3.1-BLD. Top panel: Maximum poke-induced currents observed in whole-cell mode for cells exposed to displacements up to 9.8 ± 0.8 μm (N=11; mean ± S.E.M.) and 11.8 ± 0.6 μm (N=19) above touching for WT and OSCA1.2_3.1-BLD, respectively. The percentage of cells with responses are shown above. Bottom panel: the apparent threshold in μm above touching the cell for this cohort. c, SAC I_max (top) and mmHg threshold (bottom) from WT and OSCA1.2_3.1BLD-expressing cells reveal similar activity induced by negative pressure in cell-attached patches. Data shown were obtained from the same experiments. Electrode resistances for c were similar in all cases (WT: 2.4 ± 0.1 MΩ (N=12); OSCA1.2_OSCA3.1-BLD: 2.5 ± 0.1 MΩ (N=13)).

Probing the functional role of the potential lipid-interacting residues.
a, Superposition of OSCA1.2 and OSCA3.1 around the pore fenestration. Residues of OSCA1.2 predicted to interact with lipids and corresponding residues in OSCA3.1 are shown. b, Few OSCA1.2_K435I/K536I -expressing HEK-P1KO cells respond to the poke stimulus while nearly all cells expressing OSCA1.2 WT channels respond (percentages shown above under the N of cells tested). Top panel: Maximum poke-induced currents observed in whole-cell mode for cells exposed to displacements up to 9.7 ± 1 μm (N=11; mean ± S.E.M.) and 12.1 ± 0.6 μm (N=20) above touching for WT and OSCA1.2_K435I/K536I, respectively. Bottom panel: the apparent threshold in μm above touching the cell for this cohort. c, SAC Imax (top) and mmHg threshold (bottom) from WT and OSCA1.2_K435I/K536I-expressing cells reveal no significant differences in the ability of negative pressure to activate channels in cell-attached patches (Student’s t-test). Electrode resistances were similar in all cases (WT: 2.1 ± 0.2 MΩ (N=10); K435I/K546I: 1.7 ± 0.3 MΩ (N=7)). Data were collected within 15 min of exposure of cells to high K+ used in this assay. d, Analysis of stimulus-response relationships reveal no significant differences (Student’s t-test) in pressure to half-maximal activation (P₅₀; top) or the slope of the curve (bottom). Whole cell and SAC data were obtained from cells transfected at the same time.

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