Figure 6. Requirement of phosphate groups for the lipid-dependent gating.

(a) KvAP R117C (red squares) and R120C (green triangles) in DOTAP membranes do not have specific Fv-binding. As a control, KvAP in PE/PG (black diamonds with a black dashed trend line) shows high specific Fv-binding. (b) KvAP R117H/R120H mutant channels in PE/PG membranes (0.10 mg ml⁻¹ protein instead of 0.50 mg ml⁻¹ because of the low-expression level of the mutant) have specific Fv-binding at pH 6.0, 7.4 and 8.0, but not in DOTAP membranes at all three pHs. The data at three pH levels showed the reproducibility of the binding assay even at low concentrations of channel protein. (c) In vesicles of 1:1 ratio of PE/PG: DOTAP (purple triangles; n=2, error bars are range of variation) the KvAP channels had only about 25% of the Fv-binding activity as the same quantity of channels in PE/PG (black diamonds, n=5, s.d.). The binding curve of channels in DOTAP (red squares, n=5, s.d.) is shown as a control.