Fig. 3.

KCNJ5^G151E channels have higher Na⁺ permeability than KCNJ5^G151R channels. Whole-cell currents of 293T cells transiently transfected with WT or mutant KCNJ5 channels were measured by using the patch-clamp technique and the voltage protocol indicated in A. Because incubation in solutions with physiological Na⁺ concentrations led to death of cells expressing KCNJ5^G151E, an extracellular solution containing 40 mM NaCl, 100 mM KCl, and 2 mM MgCl₂ (pH 7.4, adjusted with KOH) and an intracellular solution containing 140 mM KCl, 4 mM MgCl₂, and 5 mM Hepes (pH 7.4, adjusted with KOH) was used (control). In Ba²⁺ conditions, 1 mM Ba²⁺ was added to the above extracellular medium. In Ba²⁺/choline conditions, choline was substituted for Na⁺ as described in Materials and Methods. Representative whole-cell recordings are shown in A, and I–V curves are shown in B. In B, black, red, and green symbols denote control, Ba²⁺, and Ba²⁺/choline conditions, respectively. For better visibility, only one in two data points is plotted where curves overlap. Currents were negligible following transfection with empty vector (Fig. S3). Typical inwardly rectifying currents were observed for the WT channel; barium-sensitivity was abolished in the mutant channels. KCNJ5^G151E showed larger currents than KCNJ5^G151R, which were abolished by substitution of choline for Na⁺, indicating a predominant Na⁺ conductance. (C) Mean current amplitudes at −100 mV for WT and mutant channels using the solutions described in A. Net Na⁺ current is calculated by subtracting the current after choline substitution from the Ba²⁺ resistant current. Na⁺ current is significantly larger in the G151E mutant than in G151R mutant (P < 0.001). n ≥ 3 for each condition.