Inhibition of Kir2.1 channels by intracellular spermine and spermidine. (A) Current traces recorded from an inside-out giant patch in control and in the presence of the indicated concentrations of spermidine (spd). Currents were recorded from a holding potential of −80 mV, followed by test pulses (400 ms) in the range of −100 to +100 mV in 10-mV increments. (B) Normalized steady-state I–V_m relationships in the presence of spermine (a) and spermidine (b). Currents were normalized to that obtained at −100 mV in control. (C) Normalized G–V_m relationships shown on a semilogarithmic scale for the indicated [spermine]_i (a) and [spermidine]_i (b). Black lines show fits to Eq. 1. Fitting parameters are listed in Table S1. (D) Normalized G–V_m relationships obtained at 0.01 µM [spermine]_i (a) and 0.1 µM [spermidine]_i (b) were fit to Eq. 1. Red and green lines are the principal (high-affinity block) and minor (low-affinity block) Boltzmann components, respectively. (E) Voltage dependence of dissociation constants for high- and low-affinity block. Straight lines are fits with Eq. S2. For spermine block, K_d(0) = 0.27 µM and z = 6.7 for high-affinity block; K_d(0) = 43 µM and z = 3.0 for low-affinity block. For spermidine block, K_d(0) = 4.8 µM and z = 5.9 for high-affinity block; K_d(0) = 782 µM and z = 3.3 for low-affinity block.