Ca2+ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death

Abstract

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd⁺, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca²⁺ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca²⁺ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca²⁺ failed to induce cell death, and the Ca²⁺ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or ¹⁰Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1^-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca²⁺ overload that promotes cell death.

Keywords: Ca2+ influx; Pannexin1; cell death; hemichannel.

Fig. 1.

C-terminal truncation on hPanx1 allows increased permeability to DAPI and Etd+ but not PI. (A) Immunodetection of full-length and C-terminal truncated hPanx1 in HeLa Cx45/Panx1 double KO cells transfected with a bicistronic vector (IRES) carrying each cDNA and a reporter. α-tubulin was used as a loading control. (B) Both fl and Δ371hPanx1 HC increased their activity in response to alkaline pH (8.5) but to a greater extent the truncated isoform, n = 4. (C) Dye uptake rates from (B). (D and E) Etd+ uptake rate only increases in HeLa cells transfected with Δ371hPanx1 in response to alkaline pH, n = 3. (F) Neither full-length nor truncated hPanx1 HCs allow the flow of PI nor Sytox dyes. Data are presented as Mean ± SEM. *P ≤ 0.05; **P ≤ 0.01.

Fig. 2.

Truncated, but not full-length hPanx1 is energetically favored for the Ca²⁺ passage. (A) Electrostatic potential of full-length Panx1 hemichannel (Left, fl-hPanx1) vs. C-terminal truncated hemichannel (Right, Δ371hPanx1). (B) Free energy profile for the passage of Ca²⁺ for Panx1 HCs variants. (C) Number of permeation events for full-length (red line) and CT truncated hPanx1 HC (black line). (D) Ca²⁺ ion probability density of full-length (Left) and C-terminal truncated hPanx1 (Right). (E) Pore dimension of Panx1 hemichannel variants through HOLE software.

Fig. 3.

C-terminal truncation on hPanx1 indeed allows Ca²⁺ influx through hemichannels. (A) Cx45/Panx1 KO HeLa cells were cotransfected with fl-hPanx1 and Lck-GCaMP3 and Ca²⁺ transients were evaluated by fluorescence live cell time-lapse in response to pH 8.5. ATP was used for activating purinergic ionotropic receptors, n = 3. (B) Similarly, Lck-GCamP3 and Δ371hPanx1 were transfected, showing a rapid increase in GCamP3 signal when hemichannels were activated with alkaline pH, which was blocked with CBX, n = 3. (C and D) By using Fura-2, previous results were confirmed, showing that alkaline pH only allows an increase in Fura ratio in truncated hPanx1 transfectants, but not with fl-hPanx1 isoform. CBX partially inhibited the calcium signal evaluated with Fura-2. All recordings were made in the presence of 1 mM Na₃VO₄, a Ca²⁺ pump blocker, n = 4. (E) The area under the curve (AUC in arbitrary units: A.U.) showed a significant increase in response to alkaline pH only in Δ371hPanx1 transfectants. This response was not affected in the presence of Xestospongin C (XetC), an IP₃R antagonist but was almost completely suppressed by DCFS, n = 4. (F) There is a high correlation (R² = 0.86) between the expression level of Δ371hPanx1 and the response of hemichannels to alkaline pH recorded with Fura-2. *P ≤ 0.05, **P ≤ 0.01.

Fig. 4.

Ca²⁺ influx through truncated hPanx1 leads to cell death. (A) HeLa cells transfected with full-length hPanx1 were incubated for 4 h with Krebs pH 8.5 (lane 2), 5 µM A23187 (lane 3), or 50 µg/mL TNF-α (lane 4). Other cells were 48 h-transfected with Δ371hPanx1 vector, and proteins were extracted and loaded for western blot analysis (lane 5). (B) Densitometric analysis was performed and truncated/full-length Panx1 was plotted for each condition, n = 3. (C) DAPI uptake rates from each condition, n = 4. (D) Sytox staining (green) in HeLa cells transfected with hPanx1, varying either pH of Krebs solution or presence of 3 mM Ca²⁺ after 4 h. (E) PI staining (red) in HeLa cells transfected with Δ371hPanx1, varying either pH of Krebs solution or presence of 3 mM Ca²⁺ after 4 h. (F) Percentage Sytox staining in fl-hPanx1 was not affected by extracellular pH or presence of extracellular Ca²⁺. Consequently, this staining was not altered by cell death inhibitors, n = 3. (G) Percentage PI staining in Δ371hPanx1 transfectants changing pH, presence of extracellular Ca²⁺ and preincubation with Ferrostatin-1 (Fer-1), necrostatin-1s (Nec-1s), zVAD, or Trolox, n = 4. (H) Percentage PI staining in Δ371hPanx1 HeLa cells following a 4 h incubation with pH 7.4 or 8.5, in the absence or presence of extracellular Ca²⁺. PI staining was also plotted when different cell death inhibitors were preincubated, as depicted in legend. (I) ROS determination in Δ371hPanx1 transfectants changing extracellular pH or Ca²⁺ presence, n = 3. Data are presented as Mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 5.

C-terminal cleaved hPanx1 HCs allow Ca²⁺ influx, which favors cell death. Under exposure to alkaline pH (ALK), fl-hPanx1 HCs increase their activity allowing the influx of small molecules and dyes such as DAPI. Under apoptotic circumstances, the C-terminal tail of Panx1 is proteolyzed by caspase-3 (Casp3), increasing the radius of the pore of the hemichannel, allowing the influx of Ca²⁺ and larger dyes (DAPI and Etd⁺). Through still unexplored mechanisms, the increase in intracellular Ca²⁺ in turn favors greater cleavage of hPanx1, intracellular Ca²⁺ overload, and ultimately cell death by apoptosis.