Fig 2: Expression of rabbit Kv7.1 protein and a corresponding slow component of the delayed rectifier potassium current (IKs) in 3wRbCMs. (A) Western blot of Kv7.1 protein in freshly isolated, 48 h cultured non-transduced, and 48 h cultured GFP transduced cells. For illustrative purposes (same protein samples), the images for GAPDH and GFP are reused in Figure 6A (Kv4.3) and 6B (Kv1.4). (B) Quantification of the western blots (N = 4) shows significant downregulation of total Kv7.1 protein only in GFP-expressing cells after 48 h in culture (p < 0.05, N = 4). (C) Representative current traces before (top) and after application of IKs blocker, chromanol-293b (middle), in the presence of 5 µM E4031. Bottom traces correspond to their difference, i.e., IKs. Voltage clamp protocol is shown in the insert. (D) Cumulative I–V curve of IKs plateau measured at the end of the depolarizing pulse (N = 4, n = 12). The symbol * corresponds to p < 0.05.

Fig 3: Generation and validation of KCNQ1fs/fs_K.TET-ON hiPSC-CMs. (A) Top: Illustration of genomic KCNQ1 disruption strategy using CRISPR vectors deleting exon 3 to cause a homozygous frame shift and premature stop at the RNA level. Bottom: Schematic of genetic rescue by PiggyBac transposition-mediated integration of a doxycycline-inducible KCNQ1 transgene. (B) Top: Dox titration in KCNQ1fs/fs_K.TET-ON hiPSC-derived CMs showing dose-dependent KCNQ1 induction at RNA level (n = 3 biological replicates, **p < 0.01). Bottom: Confirmation at protein level also highlighting overall high CM differentiation efficiencies as evidenced by a-actinin staining. 1 µg/ml dox was used in subsequent experiments throughout. (C) Kv7.1 staining in a-actinin-positive cardiomyocytes showing homogeneous induction by dox and complete absence of the channel in untreated CMs. (D) Top: Under baseline conditions, dox-induced Kv7.1 on KCNQ1fs/fs background shows a predominantly perinuclear staining pattern with little abundance at the outer CM membrane marked by cadherin-2. Bottom: Endogenous Kv7.1 in 5 week-old wild-type SFS.2 CMs shows, despite faint staining, a similar subcellular localization.

Fig 4: Detection of KCNQ gene expression by immunohistochemistry. (A–E) Whole-mount preparations of guinea pig detrusor were incubated with antibodies to KCNQ1-5 subtypes. Immunoreactivity was detected in both smooth muscle bundles (SM) and in IC adjacent to and between the smooth muscle bundles for the five KCNQ subtypes tested. Inset micrographs in (A–C) show minimal immunofluorescence in pre-absorption controls for KCNQ1-3; the scale bars indicate 50 µm. (F) Micrograph of secondary antibody only control slide, showing minimal immunofluorescence. (G) Micrograph of negative control (no antibodies) demonstrating absence of autofluorescence.

Fig 5: M-currents in the hESC-RPE. (A) Representative example of the sustained currents as responses to 1000 ms voltage steps from -70 mV to 40 mV in 10 mV increments (B) and the normalized and averaged IV-curve (mean ± SEM, n = 9). (C) Current responses evoked by 1000 ms voltage pulses from -10 mV holding potential to -100 mV and up to 40 mV test potentials in 10 mV steps (D) and the normalized and averaged GV-curve (mean ± SEM, n = 5) that has been obtained using tail current analysis (see Methods). (E) The effect of 300 nM linopirdine on the currents evoked by 1000 ms voltage pulses from -70 mV to 40 mV (F) and the averaged and normalized IV-curve (mean ± SEM, n = 3). Immunostainings of paraffin-embedded vertical sections shown as xy-maximum intensity projections (apical side upwards) for KCNQ channels (gray): (G) KCNQ1, (H) KCNQ2, (I) KCNQ3, (J) KCNQ4, and (K) KCNQ5. Scale bars 10 µm. (A-F) present patch-clamp data from the cell line 11/013 and (G-K) show representative images of the cell lines 08/023 and 08/017. Abbreviations: hESC, human embryonic stem cell; RPE, retinal pigment epithelium.