Figure 4. BK_Ca channel transmembrane region interacts with Tom22.

(A) Scheme of wild type BK_Ca (1-1113) pore-forming α-subunit and different truncated proteins that were either expressed alone or co-expressed with Tom22. Numbers correspond to amino acid numbers in NCBI Accession No. U11058.2. S0-S6, transmembrane regions form the channel voltage sensor. Linker between S5-S6 forms the tetrameric channel pore region. Regulator of potassium conductance (RCK) 1 and 2 are Ca²⁺ sensors. (B) Dually labeled immunoblot for BK_Ca (top) and Tom22 (bottom) showing the ability of Tom22 to co-immunoprecipitate different BK_Ca channel regions (lanes 8-13, top). BK_Ca and Tom22 images were separated for clarity. Lanes 1-7 are controls expressing BK_Ca constructs or Tom22 alone. Squares and arrows mark the expected band sizes of each construct. Background signals (dashed squares, lanes 1-7, top) were much lower than the signals of the co-immunoprecipitated BK_Ca products (squares, lanes 8-13, top) and were subtracted to calculate Co-IP efficiency in (D). As control, the lower panel shows Tom22 successful immunoprecipitation. Tom22 was immunoprecipitated with anti-DDK mAb. Co-immunoprecipitated BK_Ca proteins were detected with anti-c-Myc pAb, and Tom22 was detected with anti-DDK mAb. (C) Control immunoblot of input lysates of cells expressing different BK_Ca regions (upper panel) and Tom22 (lower panel). BK_Ca constructs were detected with anti-c-Myc pAb and Tom22 was detected with anti-DDK mAb. Squares and arrows mark the expected band sizes. (D) Tom22 efficiency in pulling down different BK_Ca regions indicates that BK_Ca channel interacts with Tom22 mainly through the transmembrane domain (construct 1-343). Co-IP efficiency was calculated after subtracting background signals and calculating the ratio of the band intensities of co-immunoprecipitated BK_Ca constructs (i.e. B, top, lanes 8-13, squares) to corresponding immunoprecipitated Tom22 (i.e. B, lower panel, lanes 8-13), and normalized to BK_Ca construct intensities in the corresponding input lysates (i.e.. C, top, lanes 8-13). *, p<0.05 with respect to construct 1-343; #, p<0.05 with respect to the C-terminus (322-1113).