Permeant ion-dependent rectification in the IV relationship of fully activated HCN2 channels. (A and D) HCN2 TEVC currents (from a single Xenopus oocyte) obtained in response to 50-ms steps to potentials ranging from −200 to +200 mV following channel activation at −115 mV for 3 s or after a 5-ms step to −115 mV (left and right parts of sweeps in top panels and dark and light gray sweeps in bottom panels, respectively) and the difference currents (blue lines) at test potentials of +200 mV (bottom left) and −200 mV (bottom right). In this and subsequent figures, text insets indicate the Na and K concentrations in the intracellular (IN) and extracellular (OUT) solutions (see Materials and methods for estimations of internal concentrations in TEVC) while the red dashed line shows the zero current level. Sweeps were filtered at 1 kHz and sampled at 20 kHz. (B and E) Expanded views of the difference current families (from recordings shown in A and D, respectively) with −200 and +200 mV sweeps highlighted in blue. (C and F) Difference current amplitudes (determined by averaging records in B and E between 2.5 to 5 ms after the onset of the voltage step) plotted versus observed voltages. E_REV and current amplitudes with respect thereunto were determined from fits of an eighth order polynomial (thin black line). In this and subsequent IV plots, superimposed gray lines show component (dashed) and net current (solid) predictions of the GHK current equation assuming a 4:1 K:Na selectivity. (G) Expanded view around zero current of the IV plots shown in C and F. (H) Ratio of the currents recorded in elevated external K (I_K) with respect to those obtained in physiological concentrations of Na and K (I_PHYS) 150 mV negative (left) or positive (right) to E_REV. Data are mean ± SEM from five cells recorded as shown in A–G.