Abstract Background and Rationale In previous studies we found that viral (Influenza-A) lung infections decrease the expression of epithelial TREK-1 K+ channels, which was associated with worse lung injury and epithelial inflammation. However, the basic biophysical properties of alveolar epithelial TREK-1 channels remain unknown, and whether similar to our viral infection models TREK-1 channels also play a regulatory role in live bacterial infections has never been investigated. Methods We infected primary human alveolar epithelial cells (HAEC) with live Pseudomonas aeruginosa (PA) bacteria (MOI 10) for 6 hours and measured TREK-1 protein levels using ELISA assays. We quantified markers of epithelial inflammation, including ROS production and cytokine secretion (CXCL10, CCL2, IL-6) using fluorescence-based DCFDA assays and ELISA technology, respectively. To pharmacologically activate TREK-1 channels we used two structurally different agonists, BL1249 and ML335. We recorded TREK-1 currents and measured changes of the epithelial plasma membrane potential using the conventional whole-cell patch clamp technique in voltage-clamp mode at 34 °C and high-throughput Fluorescence Imaging Plate Reader (FLIPR) assays. Results We found that infection of primary HAEC with PA resulted in a 60% decrease in TREK-1 channel expression in primary HAEC. TREK-1 channel downregulation was associated with increased pro-inflammatory ROS production and secretion of inflammatory chemokines/cytokines (CXCL10, CCL2, IL-6) from HAEC. Pharmacological activation of residual TREK-1 channels in HAEC with BL1249 and ML335 counteracted the PA-induced increase in ROS production and inflammatory cytokine/chemokines secretion. Furthermore, whole-cell patch clamp recordings demonstrated that HAEC exhibit currents mediated by TREK-1 channels, as evidenced by current activation with BL1249 or ML335 and subsequent inhibition with the TREK-1 blocker PE22-28. Furthermore, TREK-1 activation counteracted PA-induced plasma membrane potential depolarization. These studies also revealed that the resting membrane potential of primary HAEC lies around -44±10mV, which is significantly depolarized compared to neurons or cardiomyocytes. Conclusion Similar to our findings in viral lung infection models, live bacterial PA infection decreases TREK-1 K+ channel expression in primary HAEC, which results in epithelial plasma membrane depolarization and increased inflammatory mediator secretion. Conversely, activation of residual TREK-1 channels repolarizes the alveolar epithelial membrane potential and protects against PA-induced inflammatory mediator secretion. Therefore, TREK-1 activation may represent a novel anti-inflammatory approach that is protective against both viral and bacterial infectious agents and may thus be a widely applicable target for future rational drug design. This abstract is funded by: NIH/NHLBI R01HL175464 (A.S.) and R01HL146821 (A.S.), DoD: W81XWH2210040 (T.Z.); AHA: 23CDA1052265 (T.Z.) DOI: https://doi.org/10.58275/AHA.23CDA1052265.pc.gr.168003, AHA 25POST1366363 (N.M.) DOI: https://doi.org/10.58275/AHA.25POST1366363.pc.gr.227247, ALA CA-1374253 (N.M.)
Angelini et al. (2026) studied this question.