Figure 4.

Relationship between presteady–state charge movement of Q457C labeled with TMR6M and the quench in fluorescence. (A) Presteady–state current records. Membrane potential was stepped for 100 ms from the holding potential (−100 mV) to a series of test values from +50 to −150 mV in 20 mV decrements. The total current I was fitted to the equation: I(t) = I₁exp(−t/τ₁) + I₂exp(−t/τ₂) + I_ss; where t is time, I₁exp(−t/τ₁) is the capacitive transient with initial value I₁ and time constant τ₁, I₂exp(−t/τ₂) is the presteady–state current of Q457C with initial value I₂ and time constant τ₂, and I_ss is the steady–state current. Q457C presteady–state current was obtained from the total current by subtraction of the capacitive and steady–state currents (6). The solid line at the left is baseline. (B) Time course of the voltage-sensitive quench of rhodamine fluorescence. Fluorescence was monitored simultaneously in the experiment of Fig. 4A. (C) Blockade of the voltage-dependent quench of rhodamine fluorescence by phlorizin. Addition of 1 mM phlorizin (Pz) to the external solution completely and reversibly abolished the voltage-dependent fluorescence quench. Phlorizin also inhibited the presteady–state currents (not shown). The time scale is the same for A, B, and C; the pulse duration is 100 ms. (D) Comparison of the Q/V and ΔF/V relations. The smooth curve was drawn according to the Boltzmann relation with Q_max = 6.5 nC, z = 1.0, and V_0.5 = −37 mV. The voltage dependence of the quench of rhodamine fluorescence was identical to that of the presteady–state charge movement. (E) Correlation between the relaxation time constants τ of presteady–state current and the quench in rhodamine fluorescence.