Intracellular Mg²⁺ blocks the ionic conductance but has no effect on cAMP binding. (A) A hyperpolarizing voltage step was used to activate the channel. Tail current was measured at 50 mV. (B) The corresponding current trace with no Mg²⁺ or 6 mM Mg²⁺. The inset shows an expanded view of the tail currents. (C) TTL signals from the charge-coupled device camera exposure port showing the timing of image collections. A series of images were collected before the voltage step (C, cAMP binding to the channels in the closed/resting state), immediately after the voltage step (V, maximal cAMP binding to channels in the open state), and during channel deactivation. (D) Raw fluorescence images collected before (C) and immediately after the voltage step (V) without Mg²⁺ in the bath solution. (E) Fluorescence images collected with 6 mM Mg²⁺ in the bath. (F) Mg²⁺ significantly inhibited both the inward macroscopic current (left, recorded at −150 mV) and outward tail current (right, recorded at 50 mV; independent samples t test: t = 6.74, P < 0.01). (G) Time course of fluorescence intensity changes without Mg²⁺ or with 6 mM Mg²⁺ in the bath. No significant difference was found (independent samples t test results at 3.65 s: t = 0.11, P = 0.99). (F and G) Error bars indicate standard error.