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. 2020 Dec 29;296:100224. doi: 10.1074/jbc.RA120.015548

Figure 5.

Figure 5

Orai1 middle transmembrane region– and cytosolic extended transmembrane region–LoF point mutations hinder the formation of an opening-permissive pore geometry.A, B, in order to determine alteration in the pore conformation, we performed cysteine cross-linking of the pore lining residues R91 and V102 for the GoF-Orai1 H134A mutant compared with different LoF–GoF Orai1 double mutants. Block diagrams exhibiting the degree of dimerization obtained via cysteine cross-linking of Orai1 R91C in comparison with Orai1 R91C H134A, Orai1 K85E R91C H134A, Orai1 R91C H134A E149K, Orai1 R91C H134A L174D, and Orai1 R91C H134A S239W (A) and Orai1 V102C in comparison with Orai1 V102C H134A, Orai1 K85E V102C H134A, and Orai1 V102A H134A E149K (B). Extent of cross-linking differed statistically significantly for the different mutants (Welch-ANOVA F(5, 10.64) = 16.08, p < 0.001 [A], F(3, 8.97) = 4.97, p < 0.05 [B]). Games–Howell post hoc test revealed significant difference for the single and triple mutants compared with the double mutant (p < 0.05). C, representative Western blots for (A) and (B), respectively. Apparently, a reduced amount of surviving cells, owing to expression of constitutively active mutants, is reflected by lighter bands. D, pore hydration profile for wildtype Orai1 and mutants. The number of water molecules is given as a function of the distance from the selectivity filter. Hydration profiles are given for wildtype in dotted black line where the gray shaded areas correspond to the standard deviation of the mean. Solid red, purple, blue, orange, and green lines correspond to Orai1 H134A, Orai1 K85E H134A, Orai1 H134A E149K, Orai1 H134A L174D, and Orai1 H134A S239W. The profiles were calculated using the last 50 ns of the simulations. Positions of the carbon α of the residues delineating the pore are given on the top axis, while the distance from the selectivity filter is given on the bottom axis. E, molecular dynamics simulations demonstrate increased pore helix rotation and pore hydration in GoF mutation H134A. Superposition of snapshots at t = 400 ns from molecular dynamics simulations of WT (gray) and H134A (red) as viewed from the top. Pairs of diagonal subunits viewed from the side (bottom). E–H, superposition of snapshots at t = 400 ns from molecular dynamics simulations of H134A (red) and K85E H134A (purple) (E), Orai1 H134A E149K (blue) (F), Orai1 H134A L174D (orange) (G), Orai1 H134A S239W (green) (H) as viewed from the top. Pairs of diagonal subunits viewed from the side (bottom). GoF, gain of function; LoF, loss of function.