Figure 3.
Figure 3. Mean extents of endogenous disulfide-bond formation between Cys substituted in the extracellular flank of S0 and Cys substituted in the extracellular flanks of S1–S6. (A) S0 flank and S1 flank; (B) S0 flank and S2 flank; (C) S0 flank and S3–S4 loop; (D) S0 flank and S5 flank; and (E) S0 flank and S6 flank. The four residues substituted by Cys in the S0 flank are indicated along the front edge of the base, and the residues substituted by Cys in the paired flank are indicated along the right edge of the base. The extent of disulfide bond formation is represented by bars in the z direction, placed at the intersection of the lines from the paired Cys-substituted residues. In the cases in which the mean extent of disulfide bond formation was zero, the value 0.5% was plotted to identify these pairs as tested. Pairs that were not tested lead to empty intersections. Except for S3–S4, the residues closest to the membrane are closest to the front, right corner of the base. For the S3–S4 loop, L199 is closest to the predicted membrane domain of S3, and S202 is closest to the predicted membrane domain of S4. (F) Proposed location of the extracellular end of S0 relative to the extracellular ends of S1–S6, consistent with the extents of disulfide bond formation. We have taken a model of the structure of Kv1.2 in the closed state (Yarov-Yarovoy et al., 2006) as a model for the structure of BK α S1–S6. In this en face view, each of the four α subunits is rendered in a distinct color. We have marked the extracellular end of each TM helix with a numbered circle in the same color as the subunit. The extracellular end of S0 is indicated as a circle marked with a zero. Its position is consistent with the cross-linking results in A-E. We have taken the means of the top three extents of cross-linking in each of the panels A–E as a measure of the relative proximity between the segments: the mean extent of cross-linking from S0 to S1 was 66 ± 20%; from S0 to S2, 52 ± 11%; from S0 to S3–S4, 96 ± 0%; from S0 to S5, 8 ± 1%; and from S0 to S6, 2 ± 1%.

Mean extents of endogenous disulfide-bond formation between Cys substituted in the extracellular flank of S0 and Cys substituted in the extracellular flanks of S1–S6. (A) S0 flank and S1 flank; (B) S0 flank and S2 flank; (C) S0 flank and S3–S4 loop; (D) S0 flank and S5 flank; and (E) S0 flank and S6 flank. The four residues substituted by Cys in the S0 flank are indicated along the front edge of the base, and the residues substituted by Cys in the paired flank are indicated along the right edge of the base. The extent of disulfide bond formation is represented by bars in the z direction, placed at the intersection of the lines from the paired Cys-substituted residues. In the cases in which the mean extent of disulfide bond formation was zero, the value 0.5% was plotted to identify these pairs as tested. Pairs that were not tested lead to empty intersections. Except for S3–S4, the residues closest to the membrane are closest to the front, right corner of the base. For the S3–S4 loop, L199 is closest to the predicted membrane domain of S3, and S202 is closest to the predicted membrane domain of S4. (F) Proposed location of the extracellular end of S0 relative to the extracellular ends of S1–S6, consistent with the extents of disulfide bond formation. We have taken a model of the structure of Kv1.2 in the closed state (Yarov-Yarovoy et al., 2006) as a model for the structure of BK α S1–S6. In this en face view, each of the four α subunits is rendered in a distinct color. We have marked the extracellular end of each TM helix with a numbered circle in the same color as the subunit. The extracellular end of S0 is indicated as a circle marked with a zero. Its position is consistent with the cross-linking results in A-E. We have taken the means of the top three extents of cross-linking in each of the panels A–E as a measure of the relative proximity between the segments: the mean extent of cross-linking from S0 to S1 was 66 ± 20%; from S0 to S2, 52 ± 11%; from S0 to S3–S4, 96 ± 0%; from S0 to S5, 8 ± 1%; and from S0 to S6, 2 ± 1%.

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