Characterization of cation currents in human lung vascular endothelial cells

Ion channels in the lung vascular endothelial cells (LVECs) are essential in lung vascular function. Ca 2+ influx through Ca 2+ -permeable cation channels and K + efflux through K + -permeable non-selective cation channels (NSCC) and voltage-gated K + (K V ) channels have been implicated in regulating cell phenotypical transition, cell senescence and inflammasome activation. Single-cell RNAseq data reveals that multiple cation channels are expressed in LVECs. In this study, we aimed to characterize non-selective cation currents ( I NSCC ) and voltage-dependent inward ( I in ) and outward ( I out ) currents in human LVECs and examine the pharmacological properties of the channels expressed in LVECs. In the whole-cell recording configuration, we were able to record voltage-dependent I in and I out in LVECs bathed in extracellular solution containing 141 mM NaCl and 1.8 mM CaCl 2 (with the intracellular pipette solution containing 135 mM KCl, 5 mM Na 2 ATP and 10 mM EGTA) with the lineal currents subtracted using P/4 leakage-current subtraction protocol. Depolarizing the cells from a holding potential of -70 mV to a series of test potentials ranging from -40 to +20 mV elicited a rapidly inactivating I in and a rapidly inactivating component followed by a sustained component of I out . Approximately 13% of tested LVECs exhibited the rapidly inactivating voltage-dependent I in and I out , while all cells showed the sustained voltage-dependent I out . The voltage-dependent I in was present at -40 mV and maximized at 0 mV, similar to Ca 2+ currents through voltage-dependent Ca 2+ channels (VDCC). All cells showed sustained voltage-dependent I out (sensitive to 4-AP), likely K + currents through K V channels. These results indicate that human LVECs functionally express VDCC as responsible for rapidly inactivating I in and K V channels for at least two components of I out (rapidly inactivating and sustained currents). In these experiments, Ca 2+ -activated K + and ATP-sensitive K + currents were minimized by including 10 mM EGTA and 5 mM ATP in the Ca 2+ -free pipette (intracellular) solution. We also recorded I NSCC , elicited by both ramp and step voltage protocols, in LVECs held at 0 mV (which inactivates voltage-dependent channels) with no P/4 leakage current subtraction. Test potentials ranging from -80 to +80 mV in 20-mV increment from the holding potential of 0 mV elicited currents that reversed at 0 mV in LVECs with the extracellular solution containing 141 mM NaCl and 1.8 mM CaCl 2 and the intracellular (pipette) solution containing 135 mM KCl, 4 mM MgCl 2 , and 5 mM Na 2 ATP. The currents at -80, 0, and +80 mV were -154.8±20.8, 0.3±5.5, and 211.7±28.8 pA (n=38 cells), respectively. Both inward and outward currents were sensitive to 2-APB (100 µM). Given the Western blot and single-cell RNAseq data showing the expression of KCNA5 and TRPC6 channels in LVECs, we also examined the biophysical and pharmacological properties of these channels in KCNA5 - and TRPC6 -transfected HEK cells. In summary, our data indicate that human LVECs express multiple voltage-dependent and voltage-independent cation channels. Ca 2+ currents are potentially generated by, at least, Ca 2+ influx through VDCC and NSCC, while K + currents are carried by K + efflux through K V channels and NSCC. This study is supported in part by a grant from the NHLBI/NIH (HL171538). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.