Fig 2: Cysteine residues in the luminal loop regulate tetrameric channel formation and channel activity. (A) IP3Rs contain an IP3-binding region in the cytoplasm, six TMDs, and four luminal cysteine residues in the vicinity of the channel pore. (B) IICR activity of each IP3R cysteine mutant. After transfection of the indicated cDNA into IP3R TKO cells, the cells were stimulated with 1 μM BK. IICR in each cell line was measured using Yellow Cameleon 3.6 (YC3.6) with a Varioskan LUX fluorescent plate reader. The average peak amplitudes of the calcium response are shown. The data present the means ± SD. *P < 0.05. (C) The fractionation of IP3R by the sucrose density gradient. After cell lysis with 1% Triton X-100, the cell lysates were fractionated by centrifugation on a 15 to 60% linear sucrose gradient. The proteins in each fraction were separated by SDS-PAGE and immunoblotted with an anti-IP3R1 antibody. Fraction nos. 7 to 9 have molecular weights corresponding to IP3R dimers, and fraction nos. 11 to 13 have molecular weights corresponding to IP3R tetramers. The red/blue lines indicate the peak fractions. (D) The dose–response curves of IICR after cell treatment with BK. The mean peak amplitudes of the YC3.6 signal (±SEM) after BK stimulation are shown. (E) The fractionation of endogenous IP3R in the indicated KO cells by sucrose density gradient. Sucrose density gradient fractionation and protein detection were performed with each KO cell under the same conditions as those described in C.