Dephosphorylation is independent of signaling events downstream of GC. (A–F) Signaling events after stimulation with 25 nM resact at different extracellular K⁺ concentrations ([K⁺]_ex). Each trace represents the mean of at least three recordings. (A) Changes in V_m detected by di-8-ANEPPS. The dashed line indicates the resting potential. (B) Changes in pH_i detected by BCECF. ΔR represents the ratio of fluorescence at 540 and 494 nm; excitation is at 425 nm. (C) Changes in [Ca²⁺]_i detected by Fluo-4. (D) Percentage of dephosphorylated GC after 3 min of stimulation with 25 nM resact at different [K⁺]_ex. (E) Apparent time constant τ of dephosphorylation at [K⁺]_ex. (F) Changes in cGMP concentration after resact stimulation at 9 and 58 mM [K⁺]_ex in the presence of 2 mM IBMX. Lines represent exponential fits. D–F represent the means ± SD of six experiments based on duplicates. (G) Resact-induced dephosphorylation in ASW or with additional 1 µM calyculin A or 1 µM okadaic acid. (H) Resact-induced dephosphorylation in ASW or with additional 10 µM sanguinarine chloride. In G and H, the amount of GC was adjusted to 120 nM, and sperm were stimulated with 250 nM resact in the presence or absence of the respective phosphatase inhibitors. Time after resact stimulation (seconds) is indicated above the respective lanes. The molecular masses of two protein size markers are shown on the left.