Abstract

Airway mucociliary clearence (MCC) is the main mechanism of lung immune innate defence, where its activity maintains the airways free of infection and obstruction. Defects in MCC are present in chronic lung diseases, such as cystic fibrosis, chronic obstructive pulmonary disease, and asthma. Adequate hydration of airways, maintenance of ciliary beating frequency (CBF) in epithelial cells and mucus homeostasis compromise MCC function, and are critically regulated by sodium absorption and chloride secretion in airway epithelial cells. The KCa3.1 potassium channel is essential for chloride secretion in the mouse intestine and its inactivation reduces the faecal water content. However, its role in respiratory epithelium is poorly understood. Therefore, we propose to elucidate the role of KCa3.1 in airway epithelial cells. Ussing chamber experiments showed reduced amiloride sensitive short-circuit current (Isc) in freshly isolated tracheas from Kca3.1-/- mice compared to wild type samples (-3.7 ± 4 vs -19.4 ± 5 μA * cm-2 respectively, p = 0.006). This effect was accompanied by a significant change in transepithelial voltage (Vte, -3.8 ± 0.8 vs. -2.0 ± 0.4 mV, p = 0.042), leaving basal Isc and transepithelial tissue resistance unaffected. The observed reduction in amiloride sensitive Isc in the Kca3.1-/- mice was not due to a downregulation of expression of ENaC subunits in epithelial cells from the mouse trachea evaluated by qRT-PCR. CBF measurements in tracheal epithelial cells were greatly increased in response to UTP in cells isolated from Kca3.1-/- mice compared to those isolated from WT. Such increase was also observed when WT cells were incubated with the KCa3.1 inhibitor TRAM-34 or the ENaC inhibitor amiloride previous to UTP stimulation. Similar results were obtained in human bronchial epithelial cells (HBEC) incubated with TRAM-34, as we observed a reduction in amiloride sensitive currents (3.1 ± 0.5 vs 1.6 ± 0.3 μA * cm-2 for control and TRAM-34, respectively). Incubation of HBEC with TRAM-34 reduced Il-4 induced goblet cell metaplasia, similar to that observed in a mouse model of chronic asthma. These results suggest an important and novel role for KCa3.1 epithelial function. Our results demonstrate that KCa3.1 inhibition reduces sodium absorption in both mouse and human epithelium. This effect is independent on changes in ENaC expression and might be explained by changes in cell membrane potential that do not favour the electrochemical gradient for sodium entry. In addition, inhibition of KCa3.1 or direct inhibition of ENaC increased CBF, which could benefit MCC. The mechanistics of such increase are being explored. Inhibition of Il-4 induced goblet cell metaplasia indicates that KCa3.1 function is not restricted to maintenance of electrochemical gradients for ion movement but is also part of the signalling of relevant cytokines. In summary, inhibition of KCa3.1 could be a therapeutic strategy for the management of inflammatory lung diseases with decreased MCC.