Figure 8.

Effect of access-channel blockers on the PTX-induced action of the Na,K-ATPase. Na,K-ATPase was equilibrated in standard buffer: 200 nM RH421, 50 mM NaCl, and PTX. Upon addition of 1 μM ATP, the enzyme turned over into the P-E₂ state to allow PTX (200 nM in [A] and 100 nM [B]) to modify the pump. (A) In the absence (a) or in the presence of 10 mM (b) and 20 mM (c) TPA⁺, the main difference of the ATP-induced fluorescence signal was an enhanced intermediate fluorescence level in the presence of the channel blocker. (B) In the absence (a) or in the presence of 5 μM (b), 10 μM (c), 15 μM (d), and 20 μM (e) Br₂-Titu³⁺, the only significant difference was the duration of the intermediate, PTX-dependent state, which was elongated by the presence of Br₂-Titu³⁺.