Panx1 acts independent of TRPV4 and may constitute a novel Ca²⁺ entry channel in HPV. (A) Representative western blot and (B) quantitative densitometric analysis show increased expression of Panx1 in HeLa cells transfected with pcDNA3.1+/c-(K)DYK vector containing Panx1 open reading frame (pcDNA3.1 Panx1 ORF) as compared to pcDNA3.1+/c-(K)DYK backbone control vector (pcDNA3.1 empty control) or non-transfected HeLa cells (HeLa). GAPDH served as loading control (n = 4 per group). Representative tracing (C) of intracellular Ca²⁺ concentration as measured by ratiometric imaging of fura-2 at 340 and 380 nm (F340/F380) and quantitative analysis of area under the curve (AUC) in HeLa cells (D). In the absence of extracellular Ca²⁺ (w/o Ca²⁺), hypoxia (1% O₂) causes a slight increase in intracellular Ca²⁺ concentration in both Panx1 overexpressing (pcDNA3.1 Panx1 ORF) and control (pcDNA3.1 empty control) cells. Following re-supplementation of extracellular Ca²⁺ (w Ca²⁺), the increase in intracellular Ca²⁺ concentration is more pronounced in pcDNA3.1 Panx1 ORF as compared to pcDNA3.1 empty control HeLa cells (n = 5 per group). (E) Group data show vasoconstrictive response to the TRPV4 agonist GSK1016790A (100 nMol/L; n = 3) in isolated perfused mouse lungs, measured as increase in pulmonary artery pressure (ΔPAP), which was unabated by the Panx1 specific inhibitory peptide ¹⁰Panx1 (800 µMol/L; n = 4). (F) Simultaneous treatment of PASMC by the TRRPV4 antagonist HC-067047 (20 µMol/L) and ¹⁰Panx1 (200 µMol/L) additively attenuated the increase in intracellular Ca²⁺ concentration in response to hypoxia as measured by ratiometric imaging of Fura-2. Data are mean ± SEM; data were analysed using Kruskal–Wallis test (B, E, F) or Mann–Whitney U-test (D); *P < 0.05; ns, not significant.