Fig. 3
Novel Kir4.1T290A mutation affects channel localization and function in patient-derived LCLs
a Projected Z-stacks of six LCLs showing the distribution of Kir4.1 in green, phalloidin to label F-actin in red and DAPI to label nucleus in blue. Scale bar, 10 μm. b Quantitative measurement of cytoplasmic and nuclear punctae normalized against the cytoplasmic space (as measured by F-actin distribution) and nuclear space (as measured by DAPI distribution) in Z-stacks. c Anti-hKir4.1 western of six LCLs showing the distribution of both monomeric and multimeric forms of the protein. Arrow indicates the expression of Kir4.1 protein against beta-actin loading control (blot insert at the bottom). −/+ and −/− indicates the nature of zygosity of unaffected parents and affected individuals. d Densitometric plots representing the relative expression Kir4.1 protein from three independent western experiments are represented as mean + SE. Data analyzed using ANOVA. e Whole-cell currents measured from healthy wild type controls and two unaffected parental controls in response to voltage step protocol from − 120 to 40 mV in presence and absence of 110 μM barium. Cells were clamped at Vm, equal to resting Vm (Vh = Vm). Histogram shows the subtraction of currents obtained with barium from whole-cell currents, which served as internal control for each experiment. Barium-sensitive current shows the contribution of Kir channels to whole-cell currents in each LCLs. Data analyzed by k independent Kruskal-Wallis test with Bonferroni correction and represented as +S.E. f Average membrane potential of LCLs from healthy control (wild type), two unaffected parents (III.11 and III. 12), and four affected (IV.2 to IV.4). Data analyzed using k independent group one-way ANOVA test with Tukey-Kramer post hoc tests. g Whole-cell patch-clamp recordings in response to voltage-steps from − 120 to 40 mV in 10 mV steps, from a holding potential of − 30 mV. Representative current traces from respective LCLs. h Current-voltage relationship is summarized within − 120 to 40 mV range. i Summary of inward currents discharges measured in response to induced K+ steps from 5 to 20 mM extracellular K+. For improved Kir specificity, Kir current discharges measured with and without barium. Data analyzed using k independent group one-way ANOVA test with Tukey-Kramer post hoc tests. Error bars represent +S.E. ** represents p < 0.001