Figure 3: The activation kinetics of coiled-coil mutant channels.
(a) Representative currents through 4R (red) and NIN (blue) mHv1/VSOP recorded at the indicated temperatures from HEK293 cells. The pulse protocol is indicated in the right. Arrows point positions of the mutations. (b) Accumulated data from 4R (red) and NIN (blue) mutant channels and the linear regression lines. Symbols used are shown in the figure. Dotted lines depict the linear regression lines. (c) Coiled-coil stoichiometry determined by Superdex 200 (GE Healthcare) size-exclusion chromatography monitored at 280 nm. For clear-size separation, we used HMT-tagged mHv1/VSOP-cc proteins in these experiments. The horizontal axis shows elution volume (ml), and dotted lines indicate the predicted elution volumes for tetrameric, trimeric, dimeric and monomeric proteins. The elution volume VE was corrected for the void elution volume (VO). Molecular weights were calculated using standard curves (Supplementary Fig. S9). Oligomeric state of the main component was 2.29±0.01 (n=3) for WT, 1.15±0.05 (n=3) for 4R and 2.35±0.05 (n=3) for NIN. (d) Temperature dependence of the CD signal recorded from mHv1/VSOP-cc WT and NIN mutant proteins at 222 nm. Signals were recorded while applying a heating (top) or cooling (bottom) temperature gradient.
