FIGURE 8.

Co-expression with Sur1 increases the sensitivity of Trpm4 to Ca²⁺. A, immunolabeling for Sur1 in His₆-Sur1-overexpressing HEK-293 (Sur1-HEK-293) cells, showing prominent expression in all cells (right panel) as compared with nontransfected HEK-293 cells (left panel). The nuclei were labeled with 4′-6-diamidino-2-phenylindole (DAPI; blue). B, immunoblots for Sur1, Trpm4, and CaM from HEK-293 cells or Sur1-HEK-293 cells transiently transfected to express Myc-Trpm4. Note that the abundance of calmodulin associated with Myc-Trpm4 (CaM Co-IP) is greater in cells co-expressing Sur1 and Trpm4 (lanes 2 and 4; replicate experiments) as compared with cells expressing Trpm4 alone (lanes 1 and 3). Sur1 was immunoblotted using total lysate, and was detected using anti-Sur1-a antibody. Myc-Trpm4 was immunoisolated using anti-Myc antibody, and immunoisolated proteins were detected using anti-Trpm4-a antibody and anti-CaM antibody. Bar graph: densitometric analysis showing the mean (±S.E.) abundance of calmodulin co-immunoprecipitated with Trpm4, normalized to the total amount of Trpm4, in the absence and presence of Sur1 co-expression; n = 6; **, p < 0.01. C, single channel recordings of inside-out patches from HEK-293 cells or Sur1-HEK-293 cells transiently transfected to express Ci-Myc-Trpm4; patches were pulled in a bath solution containing 1 μm Ca²⁺ to record base-line channel activity, after which the bath solution was changed to one containing 2 mm Ca²⁺ to maximally activate all channels. The patches were studied using the voltage clamp protocol depicted. Bar graph, mean (±S.E.) open channel probability of Trpm4, observed at +100 mV and at −100 mV, in 1 μm Ca²⁺ relative to that in 2 mm Ca²⁺, in cells expressing Trpm4 alone or co-expressing Sur1 and Trpm4. Charge carrier, Cs⁺; **, p < 0.01.