Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C94
Genetic inactivation of the KCNN4 K+ channel protects from lethality in a cystic fibrosis mouse model
C. A. Flores1, L. P. Cid1, F. V. Sepulveda1
1. Centro de Estudios Cientificos, Valdivia, Chile.
Cystic fibrosis (CF) is produced by mutations in the CFTR Cl- channel gene that dramatically reduce epithelial electrolytes and fluid secretion and maintenance of a hydrated mucosa. Mouse models are useful to study the intestinal complications of CF. Electrogenic Cl- secretion is strictly coupled to the activity of basolateral cAMP- and Ca2+ -dependent K+ conductances. We have demonstrated that KCNN4 corresponds to the Ca2+ -activated K+ conductance in the basolateral side of intestinal epithelium and that, its genetic silencing in mice abolishes Ca2+-activated Cl- secretion and reduces water content of the stools (Flores et al., 2007). The Ca2+-dependent Cl- secretion in the intestine of CF mice has been postulated as an important factor that increases survival of these animals. Given the role of KCNN4 in regulating secretion we investigated if the abolition of KCNN4 activity in a mouse model of CF can modify the intestinal phenotype of the disease. Control (WT), KCNN4 KO and mutant CFTR-ΔF508 (CF) animals were used. Breeding of double mutant animals was done by mating of double heterozygous. Survival and weight progression curves for the offspring were constructed. After killing, dissection of the intestine was performed to obtain tissue samples for Periodic Schiff Acid (PAS) staining. Electrogenic secretion of the colon was studied by Ussing chamber technique. Water content of the faeces was also measured. Genetic silencing of KCNN4 reduced the lethality of CF mice from 43% to 3% after 60 days (13 and 3 dead out of 30 mice on each group respectively). The mean body weight of CF animals at 40 days of age was 10.8 ± 0.8 g, almost half of the weight recorded in WT and KCNN4 KO animals (19.8 ± 0.8 and 19.2 ± 1.2 g respectively). The double mutant did not show any improvement in weight (10.7 ± 0.7 g) compared to CF. Electrogenic cAMP- and calcium-activated currents were absent in colon from both CF and double mutant animals. Water content of stools was reduced in the KCNN4 KO (46 ± 1.6 %) with respect to WT (57 ± 0.8 %), CF (51 ± 2 %) and double mutant (53 ± 2 %) animals. PAS staining demonstrated that both CF and double mutant animals had and increased accumulation of mucus in the intestinal crypts. Our results show that genetic silencing of KCNN4 channels greatly improved the survival of animals bearing mutant CFTR channels. The observed reduction in lethality does not seem to be related to improvement of the intestinal transport function since double mutant mice did not show alternative chloride conductances, changes in water content in faeces, reduced accumulation of thick mucus or increased body weight when compared to CF animals. Since KCNN4 is broadly expressed in mammalian tissues we now aim at studying the role of KCNN4 in non epithelial cells that could be pathologically affected in the CF.
Where applicable, experiments conform with Society ethical requirements