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

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 ⁺ (K_Ca) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) K_Ca, which may in turn amplify Ca²⁺ influx by increasing the electrochemical Ca²⁺ gradient and thus, promote lung injury. We therefore hypothesized that endothelial K_Ca channels may contribute to the progression of TRPV4-mediated VILI.

Male C57Bl/6J mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the non-selective K_Ca 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 K_Ca channels. Analogously, changes in intracellular potassium concentration ([K⁺]_i) and membrane potential (V_m) were imaged in vitro.

Endothelial TRPV4 deficiency or inhibition of K_Ca 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 K_Ca 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.

K_Ca 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.