Pannexins form gap junctions with electrophysiological and pharmacological properties distinct from connexins

Abstract

Stable expression of pannexin 1 (Panx1) and pannexin 3 (Panx3) resulted in functional gap junctions (GJs) in HeLa cells, but not in Neuro-2a (N2a) or PC-12 cells. The glycosylation pattern of expressed Panx1 varied greatly among different cell lines. In contrast to connexin (Cx) containing GJs (Cx-GJs), junctional conductance (Gj) of pannexin GJs (Panx-GJs) is very less sensitive to junctional voltage. Both Panx1 and Panx3 junctions favoured anionic dyes over cations to permeate. Though, carbenoxolone (CBX) and probenecid blocked Panx1 hemichannel activity, they had no effect on Panx1-GJs or Panx3-GJs. Extracellular loop 1 (E1) of Panx1 possibly bears the binding pocket. The Cx-GJ blocker heptanol blocked neither Panx1 hemichannel nor Panx-GJs. Unlike the GJs formed by most Cxs, CO2 did not uncouple Panx-GJs completely. Oxygen and glucose deprivation (OGD) caused lesser uncoupling of Panx-GJs compared to Cx43-GJs. These findings demonstrate properties of Panx-GJs that are distinctly different from Cx-GJs.

Figure 1. Panx1 and Panx3 form functional gap junctions (GJs) in HeLa cell.

The cartoon diagram represents recording of junctional currents using dual patch clamp. Series of voltage (V₁) steps were applied to cell-1, keeping the voltage of cell-2 (V₂) constant at 0 mV. Junctional current (I_j) corresponds to the current recorded from cell-2. Representative I_j traces are depicted in (A, B and C). Panx1 and Panx3 (A and B) transfected cells exhibited significant I_j. (C) non-transfected control HeLa cells showed much less I_j, compared to Panx1 and Panx3 expressing cells. (D and E) V_j-G_j(ss) plots of Panx1 and Panx3 compared with that of Cx43. G_j(ss) of both Panx1 and Panx3 remained relatively unchanged with increasing V_j. G_j(ss) of Cx43 showed voltage dependence in similar experimental conditions. Values are the mean ± SEM of 4–11 independent experiments. The dashed line in the Cx43 plot represents the fitted curve with the two states Boltzmann equation. (F) V_j-G_j(ss) plot of endogenous GJ, recorded from non-transfected wild type (WT) HeLa cells (n = 7). The sharp decrease of G_j(ss) with increasing V_j resembles the V_j-G_j relation of Cx45-GJs.

Figure 2. Differential glycosylation pattern of Panx1.

(A) Representative western blots of Panx1-eGFP, expressed in N2a, HeLa and PC-12 cells. 50 μg of total proteins were resolved on 7.5% SDS-polyacrylamide gel. The protein was transferred to PVD membrane and probed with anti-GFP antibody. The higher molecular weight band corresponds to the glycosylated form. (B) The glycosylated Panx1 band disappeared upon treatment with 10 units of PNGase-F for 30 minutes. Corresponding bands of Panx1 (A and B) are cropped from the full blots and presented. Picture of the full-length blots are shown in supplementary section. (C) Comparison of the glycosylated fraction of Panx1 in different cell lines. In N2a and PC-12 cells, 43 ± 3% and 79 ± 3% of total Panx1 are glycosylated, whereas, in HeLa cells, glycosylated fraction represents only 8 ± 2% of total Panx1. Band intensity was quantified with Image J software. Values are the mean ± SEM of seven experiments.

Figure 3. Pannexin gap junctions are permeable to anionic but not to cationic dyes.

Passage of fluorescent dyes through (A) Panx1 and (B) Panx3 GJs. Dye was injected in one cell of a pair (donor cell; Cell-1) through the patch pipette and its passage to the recipient cell (Cell-2) was monitored. In 5 minutes time, anionic Alxa 350 reached to the recipient cell both through Panx1 and Panx3 GJs. None of the cationic dyes (e.g. PI, EtBr and DAPI) passed through Panx1 and Panx3 GJs, indicating the cells are not connected by cytoplasmic bridges. Dyes were not detected in the recipient cells even after 10 minutes. Cell-cell coupling was confirmed in every cell pair by measuring G_j at the end of the experiment. (C) As a positive control, Cx43-GJs expressed in HeLa cells showed the permeation of both anionic and cationic dyes. ‘BF' represents bright field image.

Figure 4. Pannexin gap junctions are insensitive to CBX, probenecid and heptanol.

(A) Representative V_j protocol- and I_j recorded from a Panx1 stably transfected cell pair. I_j was not inhibited in presence of 30 μM CBX. Solid bar over the current trace represents duration of CBX application. (B) Effect of different blockers on G_j of Panx and Cx43 junctions. G_j was measured at +40 mV of V_j. CBX and probenecid (2 mM) did not change the G_j of Panx-GJs in 5 minutes. G_j of Cx43 was reduced by 30–42% with CBX and probenecid, respectively. 2 mM heptanol completely uncoupled Cx43 junctions within 1–2 minutes. However heptanol had no effect on Panx-GJs. Data are the mean ± SEM of 6-11 independent experiments.

Figure 5. CBX and probenecid have no effect on chimeric Panx1 hemichannels, bearing the first extracellular loop (E1) of Cx43.

(A) I-V curve generated from mock (peGFPN1) transfected CHO cells, did not show Panx1 type currents. (B) I-V curve of Panx1, recorded from transfected CHO cells. Panx1 hemichannel currents were inhibited promptly (within 1–2 min) by 30 μM CBX (red line) and 2 mM probenecid (blue line). (C) Panx1-E1-Cx43 exhibited similar I-V characteristics as wild type Panx1, but was not inhibited either by CBX or by probenecid. (D) I-V curve generated from Panx1-E2-Cx43 did not show significant currents.

Figure 6. Acidosis and Oxygen glucose deprivation (OGD) uncoupled Pannnexin junctions to a lesser extent than Cx43 junction.

Acidosis was achieved by saturating external solution with 100% CO₂. (A) Representative junctional current traces showing the effect of CO₂ on Panx1-GJs and Cx43-GJs. Although CO₂ uncoupled Cx43 junction rapidly it had little inhibitory effect on Panx junctions. Solid lines over the current traces represent duration of CO₂ application. (B) Inhibition of G_j by CO₂ and OGD. OGD was achieved as described in the method section. Both Panx1-GJs and Panx3-GJs were inhibited by CO₂ and OGD to much lesser extent, compared to Cx43-GJs. Values are the mean ± SEM of 7-11 experiments.