Hereditary mutations of KCNQ1 K+ channels can lead to the cardiac long QT syndromes, Jervell and Lange-Nielson (JLNS) or Romano-Ward (RWS) syndromes. KCNQ1 is known to mediate cardiac slowly activating delayed rectifier current (IKs) although it is not clear how mutations in the channel decrease channel function. The aim of this study was to investigate whether the intracellular retention of mutant KCNQ1 channels in the endoplasmic reticulum (ER) is a feasible mechanism to explain the decreased KCNQ1 channel function observed in JLNS/RWS.
Enhanced green fluorescent protein (EGFP) was cloned onto the C-terminus of the long isoform of KCNQ1 and transfected into CHO-K1 cells with or without the accessory subunit, IsK. ER localisation was determined by co-transfection of pDsRed2-ER (an ER-retained red fluorescent protein, Clontech, UK). Images were acquired with a Bio-Rad Radiance 2000 scanning laser confocal microscope. Co-localisation of green and red fluorescence was quantified using LaserPix software (Bio-Rad, UK). Due to variability of transfection efficiency, data are compared to controls recorded in parallel. Data are expressed as means ± S.E.M. Statistical analysis was performed using Student’s unpaired t test or one-way ANOVA, as appropriate.
Expression of wild-type KCNQ1 resulted in 37.3 ± 2 % (n = 44) of the protein being retained in the ER. Co-expression of IsK significantly reduced this to 27.0 ± 2 % (n = 47, P < 0.05). When mutant channels were expressed with IsK, of six mutants screened, R243H, E261D, R518X, Q530X, 1008delC and R594Q, all showed significantly increased ER retention compared to KCNQ1+IsK (range 46-53 %, n = 15-26, P < 0.001). A seventh mutant, L273F, also showed increased ER retention, although this was not significantly different from the KCNQ1+IsK control (36.2 ± 4 %, P < 0.05, n = 28). Incubation of cells at 27 °C for 24 h which has previously been shown to recover trafficking of some HERG channel mutants (Zhou et al. 1999), did not significantly reduce ER colocalisation of any of the mutants tested (n = 6-14). In other experiments, 24 h incubation of channel-modulating drugs had no significant effect on retention of R243H channels being 38.0 ± 4 %, 43.3 ± 3 % and 37.2 ± 5 % ER retained in the presence of HMR1556 (100 nM), chromanol 293B (10 µM) and mefenamic acid (100 µM), respectively (control, 38.1 ± 1.0 %, n = 6-15).
ER retention of KCNQ1 channels is a potential mechanism by which mutations could cause JLNS/RWS. It is not known if this reflects a defect in trafficking ability per se, or whether mutations abrogate the ability of the channel protein to interact with IsK. Unlike some mutant HERG channels, KCNQ1 trafficking ability does not appear to be sensitive to either temperature or the presence of pharmacological channel modulators.