Whole-cell K⁺ currents of WT hK_v7.1 and mutant channels (E290K and E290A) expressed in CHO cells. (A) Schematic diagram of the structure model of K_v7.1 in the open state (Smith et al., 2007), showing the residue E290 in red. Amino acid sequence alignment from residues 284–295 between human and mouse K_v7.1 is shown in the middle. The E290 is located in the external S5-pore linker. (B) Green fluorescence protein (GFP)-alone transfected cells did not yield measurable currents. (C) Examples of K⁺ current traces recorded from CHO cells after 24–48 h of transfection with WT hK_v7.1 elicited from a holding potential of −70 mV using a family of test pulses ranging from −90 to 60 mV for 2.5 s with a 10-mV increment followed by a voltage step to −30 mV. (D and E) Neither E290K nor E290A yielded measurable current after 24–96 h of transfection using the pulse protocol described in C. The point mutations converted residue 290 from negative to positive and nonpolar neutral amino acids, respectively. (F) The corresponding current density–voltage relationships (n = 17). The averaged current density from the last 50 ms of the test pulse was used. (G) The reversal potential of the K⁺ current was determined in each experiment using an instantaneous tail current protocol elicited from a holding potential of −70 mV by applying a prepulse of 40 mV for 1.5 s before stepping to a family of test potentials from −110 to −10 mV (10-mV increments) and analyzing the resulting deactivation currents. The current and voltage-clamp protocol (inset) are shown on the left and the corresponding instantaneous current–voltage relationship is shown on the right for 4 mM K⁺_e.