Functional studies of human K+ channels KCNH1 and KCNK4 and their role in human pathogenesis

Potassium (K+) channels constitute the most diversified class of ion channels with regard to structure and gating characteristics. They contribute to the maintenance and stabilization of the resting potential and are key players in regulating cell excitability and functions in response to multiple signals (Tian et al., 2014). In recent years, the aberrant function of some of these channels has been documented to affect development and underlie syndromic disorders (Bauer et al., 2018). KCNH1 (MIM 603305) is a member of the EAG (ether-à-go-go) family of voltage-gated K+ channels (Cázares-Ordoñez and Pardo, 2017). Recent studies have demonstrated that gain-of-function mutations in KCHN1 are implicated in Zimmermann-Laband syndromes (ZLS; MIM 135500;Kortüm et al., 2015) and other forms of developmental deficits that include mental retardation and epilepsy (Simons et al., 2015;Bramswig et al., 2015; Mégarbané et al., 2016; Fukai et al., 2016). These findings suggest that KCNH1 might be important for cognitive development in human. Recently, gain-of-function mutations in KCNK4 (MIM 605720), encoding a two-pore-domain K+ channel (K2P), have been reported in subjects with a phenotype of facial dysmorphism, hypertrichosis, epilepsy, developmental delay/ID, and gingival overgrowth (FHEIG syndrome, MIM 618381; Bauer et al., 2018). The clinical features of the KCNK4-related condition are reminiscent of ZLS (Kortüm et al., 2015), providing evidence for a channelopathy caused by hyperactivation of K+ channels, including KCNH1 and KCNK4. Some ion channels are bifunctional proteins contributing to several cell functions (Hegle et al., 2006). To date, only electrophysiology experiments were performed to explore the mechanism involving KCNH1 and KCNK4 in ZLS and FHEIG related developmental processes (Kortüm et al., 2015; Simons et al.,2015; Bauer et al., 2018). Based on these considerations, major aims of my PhD project were to better characterize the cell role of KCNH1 and KCNK4 channels and to explore the functional impact of the KCNH1-ZLS and KCNK4-FHEIG mutations using cellular and molecular biology approaches, including Immunofluorescence, Western Blot and Real-Time Quantitative PCR. To this aim, we used cell lines, control fibroblasts and primary skin fibroblasts derived from ZLS-patients carrying mutations in KCNH1 (p.Arg330Gln and p.Leu352Val) and from FHEIG-patients carrying mutations in KCNK4 (p.Ala172Glu and p.Ala244Pro). We demonstrated impaired proliferation of KCNH1 and KCNK4 mutant fibroblasts, confirming the role of KCNH1 as a regulator of cell cycle of non-transformed cells and highlight a new function for KCNK4 in regulation of cell proliferation. We also found a significant increase of cilia number and a cilium-related pathway, i.e. SHH pathway, activation in all the mutant fibroblasts, thus suggesting functional role of KCNH1 and KCNK4 in cilia regulation. Moreover, confocal microscopy analysis revealed defects in cilia morphology in fibroblasts from ZLS patients. Confocal analysis refined also the reported KCNH1 localization in the cilium (Sánchez et al., 2016) to be concentrated at the centrosome and ciliary pocket regions for both wild-type and mutant fibroblasts. Finally, immunofluorescence analysis conducted in 3 wild-type fibroblasts and cells lines disclosed a nucleolar localization of KCNK4 channels, possibly indicating unrevealed roles of KCNK4. In summary, we demonstrated that KCNH1 and KCNK4 may have specific ion channel-independent function converging on some share cellular pathways whose alteration might affect development processes. Overall, these findings highlight the importance to characterize the intracellular role of K+ to shed light on the mechanisms of pathogenesis of ZLS and ZLS related developmental processes.