Connexin 43 hemichannels are permeable to ATP

Abstract

Astrocytes are electrically nonexcitable cells that communicate by means of Ca(2+) signaling. Long-distance intercellular Ca(2+) waves are initiated by release of ATP and activation of purinergic receptors on nearby cells. Previous studies have implicated connexin 43 (Cx43) in ATP release, but definitive proof that ATP exits through Cx43 hemichannels does not exist. Here, through several alternative approaches, we show that ATP anions can permeate through Cx43 hemichannels. First, openings of Cx43 hemichannels were detected in both cell-attached and inside-out patch recordings in C6 cells expressing Cx43, but not in C6 cells expressing Cx43-eGFP (enhanced green fluorescent protein) or a C-terminus truncation mutant of Cx43. Second, Cx43 hemichannel openings were inhibited by three structurally different gap-junction channel blockers, but not by the P2X(7) blocker Brilliant blue G. Third, bioluminescence imaging of ATP combined with single-channel recording in the inside-out patch configuration showed that ATP efflux coincided with channel openings and was absent when the Cx43 hemichannel was closed. Fourth, ion replacement experiments confirmed that Cx43 hemichannels are permeable to ATP. In summary, these observations provide the first direct evidence for efflux of ATP through Cx43 hemichannels. Furthermore, a putative Cx43 hemichannel with characteristics identical to the Cx43 hemichannel in C6 cells was identified in the membrane of hippocampal astrocytes in acutely prepared slices.

Figure 1.

Cx43 hemichannels in cell-attached patches from Cx43-expressing C6 cells. A, Cell-attached patch recording with the pipette solution containing 130 mm Na₂ATP (top trace) or NaCl (bottom traces) and 20 mm TEA showed the opening (o) and closing (c) states of Cx43 hemichannels. Voltage steps of −160 to 0 mV were applied to the patch pipette. V_m that was the difference between a voltage step and the RMP was presented at the left of each trace. A substate of long openings with slow transitions was also observed (o_sub and arrows). B, The I–V relationship for cell-attached recordings with NaCl (open circle, n = 5 patches) or Na₂ATP (closed circle, n = 6 patches). C, The I–V current of Cx43 hemichannels with MgATP (MgATP) in the patch pipette. D, Open and closed time distributions for a Cx43 hemichannel with a Na₂ATP pipette at holding potential of 70 mV. The decay constant (τ) for open and closed time was calculated by exponential regression with one and two constants, respectively. The numbers indicate the mean decay constants (τ). E, The mean open time constant at different holding potentials. The open time of Cx43 hemichannels is rectified. n = 6 channels. F, Inside-out patch recording from a representative inside-out patch showed fast-gating openings (o_f) and slow-gating openings (o_s). A subconductance of slow-gating openings (o_sub) was also observed. The inside-out patch was excised from the cell-attached patch configuration into the artificial CSF solution. V_m across patches was +80 mV. G, The I–V relationship for inside-out recordings with voltage steps. Error bars indicate SEM.

Figure 2.

The recorded channel is a Cx43 hemichannel. A, Fast-gating openings of Cx43 hemichannels in cell-attached patches from a Cx43-expressing C6 cell (Cx43⁺), a control Cx43-deficient cell (Cx43⁻) that does not express Cx43, a Cx43-eGFP-expressing C6 cell (Cx43-GFP), and a truncated Cx43-expressing C6 cell (Cx43-M257). B, The number of Cx43 hemichannels per cell-attached patch. C, Twenty-six picosiemen openings of Cx 43 hemichannels in inside-out patches from Cx43⁺, Cx43⁻, Cx43-eGFP, and Cx43-M257 cells. D, The mean number of 26 pS Cx43 hemichannels per inside-out patch. **p < 0.01 compared with Cx43⁺, Student's t test. E, Immunolabeling against Cx43 (white) in Cx43⁺, Cx43⁻, and Cx43-M257 C6 cells and immunolabeling against eGFP in Cx43-eGFP C6 cells (Cx43-GFP). Scale bars, 10 μm. Error bars indicate SEM.

Figure 3.

Blockade of Cx43 hemichannels by gap-junction and anion channel blockers. A, Inside-out patch recording of 26 pS Cx43 hemichannels during control, 6 s, and 24 s after application of gadolinium (Gd⁺³, 25 μm), carbenoxolone (100 μm), and NPPB (100 μm). B, Mean data showing the time course of the P_o of 26 pS Cx43 hemichannels during control and application of Gd⁺³, carbenoxolone, and NPPB. P_o was calculated for each channel during 2 s using the Clampfit program of pClamp 9.0. p < 0.001, one-way ANOVA; n = 11, 12, and 9 patches for Gd⁺³, carbenoxolone, and NPPB, respectively. C, Cell-attached patch recording (top trace) and inside-out patch recording (bottom trace) in the presence of Brilliant blue G (BBG, 1 μm) in the patch pipette. D, Percentage of cell-attached and inside-out patches that showed 165 pS fast-gating openings (cell-attached patches) or 26 pS substate openings (inside-out patches). Data from recordings with pipettes containing Na₂ATP (Con), Brilliant blue G and Na₂ATP (BBG), NaCl (NaCl), or Brilliant blue G and NaCl (NaCl/BBG) were presented. Error bars indicate SEM.

Figure 4.

Detection of ATP influx through a single Cx43 hemichannel by luciferace/luciferine. A, An inside-out patch (DIC, p1) was excised from Cx43-expressing C6 cells. The pipette solution contained 130 mm Na₂ATP. A baseline image was first obtained before the patch electrode was moved close to the puff pipette (DIC, p2) containing luciferase and luciferin (K salt, 1:3). A low pressure was applied to the puff pipette to eject the enzyme solution. When a voltage step of +80 mV (intracellular) was applied to the patch, channel openings were observed under voltage-clamp of the inside-out patch (traces over the images). Simultaneous imaging showed that light intensity increased with the channel activity. Channel openings declined after a few minutes for unknown reasons. B, A control inside-out patch without channel activity showed no light increase. C, The relationship between the increase in light intensity (ΔF/F, filled circles; F is the baseline) and the channel open probability (P_o, filled squares) with patches containing channels. No light was emitted when the patch contained no active channels (open circles). The termination of channel activity preceded the termination of light increase, which reflects that flow of the solution takes time to remove released ATP. n = 6 and 11 patches for the channel and no channel groups, respectively. Error bars indicate SEM.

Figure 5.

Blockade of ATP influx by negative holding or hemichannel blockers. A, Light induced by ATP influx when an inside-out patch was held at −80 mV (V_h = −80 mV) and +80 mV (V_h = +80 mV). Recording traces at the top of each panel illustrate hemichannel activities at open (o) and closed (c) states. The arrow (p) indicates the pipette containing the enzyme that delivered by a light, but sustained pressure. B, ATP influx when gap-junction blockers, Gd³⁺ (30 μm), carbenoxolone (Carb, 100 μm), and NPPB (100 mm) were applied in the bath solution. Patches showing hemichannel activity before action of blockers (control) were chosen to move to the enzyme pipette. The inside-out patch was held at +80 mV. C, Relative light intensity (ΔF/F) against time during holding at −80 mV (filled circles, V_h = −80 mV) and +80 mV (filled circles, V_h = +80 mV; n = 7 patches) or in the presence of gap-junction blockers (open squares; n = 9 patches; two-way ANOVA, p < 0.01 compared with absence of blockers). Error bars indicate SEM.

Figure 6.

Permeability of Cx43 hemichannels. A, Twenty-six picosiemen openings in an inside-out patch at different holding potentials (left numbers). B, Top trace, Cell-attached patch recording with the patch pipette solution containing Na₂ATP. The cell was whole-cell clamped with a pipette containing 280 mm sucrose (NaATP/Sucr). Middle trace, Both cell-attached and whole-cell pipettes contained sucrose (Sucr/Sucr). Bottom trace, Single-cell-attached patch recording with the pipette solution containing sucrose (Sucr/Cyto). C, I–V relationships with extracellular Na₂ATP and intracellular sucrose (NaATP/Sucr) or extracellular sucrose and intracellular cytoplasm (Sucr/Cyto). D, Reversal potentials (V_Rev) from cell-attached patch (cell-attached) and inside-out patch recordings (inside-out) with equivalent intracellular and extracellular NaCl (NaCl/NaCl), intracellular sucrose and extracellular Na₂ATP (NaATP/Sucr), or extracellular NaCl (NaCl/Sucr), extracellular KCl (KCl/Sucr), and extracellular Na₂ATP with cytoplasm (NaATP/Cyto). Error bars indicate SEM.

Figure 7.

Putative Cx43 hemichannels in CA1 hippocampal astrocytes. A, Putative Cx43 hemichannels in a cell-attached patch obtained from an astrocyte in an acute hippocampal slice. B, The I–V current for the channel in the cell-attached patch in A. C, Putative Cx43 hemichannels in an inside-out patch obtained from an astrocyte in a slice. D, The I–V current for the channel in the inside-out patch in C. The V_Rev is close to zero, similar to that of Cx43 hemichannels from Cx43-expressing C6 cells.