KCNC3^R423H causes dominant electrophysiological and trafficking effects on KCNC3^WT.

(A) Representative currents evoked by a step from −70 mV to +70 mV in CHO cells expressing KCNC3^WT-Clover or KCNC3^R423H-Clover, and in those transfected with both constructs KCNC3^WT-Clover:KCNC3^R423H-mRuby2 in a 1:1 ratio. (B) Mean current densities recorded in CHO cells expressing either wild-type KCNC3^WT-Clover (n = 7) or KCNC3^R423H-Clover (n = 5) and in those expressing both KCNC3^WT-Clover: KCNC3^R423H-mRuby2 (1:1) constructs (n = 6). Current density was calculated by dividing the peak current evoked by a step from −70 to +70 mV by cell capacitance. Values are shown as mean±SEM, and significance was tested using a one-way ANOVA. (C) Current-voltage relations for cells in the three conditions shown in (A) and (B). Confocal fluorescence microscopy of cells expressing KCNC3^WT-Clover (D) or KCNC3^R423H-mRuby2 (G) individually, with no channel bleed-through (E,F). (H-S) Confocal fluorescence microscopy of cells co-expressing KCNC3^WT-Clover and KCNC3^R423H-mRuby2 at ratios of 1:1 to 6:1 (KCNC3^WT:KCNC3^R423H) showing co-localization and intracellular retention of both proteins, even at the highest concentration of KCNC3^WT. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.