Calcium-activated potassium channels as amplifiers of TRPV4-mediated pulmonary edema formation in male mice.

BACKGROUND: As a mechanosensitive cation channel and key regulator of vascular barrier function, endothelial transient receptor potential vanilloid-type 4 (TRPV4) contributes critically to ventilator-induced lung injury (VILI) and edema formation. Ca2+ influx via TRPV4 can activate Ca2+-activated K + (KCa) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) KCa, which may in turn amplify Ca2+ influx by increasing the electrochemical Ca2+ gradient and thus, promote lung injury. We therefore hypothesized that endothelial KCa channels may contribute to the progression of TRPV4-mediated VILI. METHODS: Male C57Bl/6J mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the non-selective KCa antagonists apamin, charybdotoxin, or the selective IK1 antagonist TRAM34. Lung injury was similarly assessed in overventilated, endothelial-specific TRPV4-deficient mice or TRAM34-treated C57Bl/6J mice challenged with intratracheal acid installation. Changes in endothelial Ca2+ concentration ([Ca2+]i) were monitored by real-time imaging in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells (HPMECs) in response to TRPV4 activation with or without inhibition of KCa channels. Analogously, changes in intracellular potassium concentration ([K+]i) and membrane potential (Vm) were imaged in vitro. RESULTS: Endothelial TRPV4 deficiency or inhibition of KCa channels, and most prominently inhibition of IK1 by TRAM34 attenuated VILI as demonstrated by reduced lung edema, protein leak, and by quantitative lung histology. All KCa antagonists reduced the [Ca2+]i response to mechanical stimulation or direct TRPV4 activation in isolated lungs. TRAM34 and charybdotoxin, yet not apamin prevented TRPV4-induced K+ efflux and membrane hyperpolarization in HPMECs. TRAM34 also attenuated the TRPV4 agonist-induced Ca2+ influx in vitro and reduced acid-induced lung injury in vivo. CONCLUSIONS: KCa channels, specifically IK1, act as amplifiers of TRPV4-mediated Ca2+ influx and establish a detrimental feedback that promotes barrier failure and drives the progression of VILI.