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KCNC3R423H causes dominant electrophysiological and trafficking effects on KCNC3WT.

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posted on 2017-05-03, 17:26 authored by Swati Khare, Jerelyn A. Nick, Yalan Zhang, Kira Galeano, Brittany Butler, Habibeh Khoshbouei, Sruti Rayaprolu, Tyisha Hathorn, Laura P. W. Ranum, Lisa Smithson, Todd E. Golde, Martin Paucar, Richard Morse, Michael Raff, Julie Simon, Magnus Nordenskjöld, Karin Wirdefeldt, Diego E. Rincon-Limas, Jada Lewis, Leonard K. Kaczmarek, Pedro Fernandez-Funez, Harry S. Nick, Michael F. Waters

(A) Representative currents evoked by a step from −70 mV to +70 mV in CHO cells expressing KCNC3WT-Clover or KCNC3R423H-Clover, and in those transfected with both constructs KCNC3WT-Clover:KCNC3R423H-mRuby2 in a 1:1 ratio. (B) Mean current densities recorded in CHO cells expressing either wild-type KCNC3WT-Clover (n = 7) or KCNC3R423H-Clover (n = 5) and in those expressing both KCNC3WT-Clover: KCNC3R423H-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 KCNC3WT-Clover (D) or KCNC3R423H-mRuby2 (G) individually, with no channel bleed-through (E,F). (H-S) Confocal fluorescence microscopy of cells co-expressing KCNC3WT-Clover and KCNC3R423H-mRuby2 at ratios of 1:1 to 6:1 (KCNC3WT:KCNC3R423H) showing co-localization and intracellular retention of both proteins, even at the highest concentration of KCNC3WT. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.