Figure 4: Temperature-dependent activation of linker mutant channels.

(a) Amino-acid sequence of linker residues between the coiled-coil domain and the last transmembrane domain (S4) (upper panel). Three residues, VKT, mutated to GGG/AAA in (b) are underlined. Representative currents through Tandem (blue) and ΔC-Tandem (red) channels recorded at 25 °C from HEK293 cells (lower panel). Accumulated data from the Tandem (blue) and the ΔC-Tandem (red) channels and the linear regression line (right panel) are shown; the dotted lines depict the linear regression lines for the data from WT (black) and ΔC (green) channels. (b) Accumulated data from the GGG (red) and AAA (blue) mutant channels and their linear regression lines; the dotted lines are the same as in panel (a). (c) Accumulated data from the GCN4 chimeric channel with a long linker (relax) (red) or with no linker (blue) and their linear regression lines; the dotted lines are the same as in panel (a). (d) Oligomeric state analysis of the mutant channels determined by crosslinker analysis with western blotting. mHv1/VSOP WT, the GGG and AAA mutants, and the relax and no linker chimeras were expressed in HEK293 cell and treated with or without 1 mM DSS. Proteins were separated on SDS–PAGE and immunoblotted with anti-mHv1/VSOP antibody.