The ATP permeability of pannexin 1 channels in a heterologous system and in mammalian taste cells is dispensable

Abstract

Afferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly. This inconsistency could indicate that the cellular context specifies Panx1 functioning. We cloned Panx1 from murine taste tissue, and heterologously expressed it in three different cell lines: HEK-293, CHO and neuroblastoma SK-N-SH cells. In all three cell lines, Panx1 transfection yielded outwardly rectifying anion channels that exhibited fast gating and negligible permeability to anions exceeding 250 Da. Despite expression of Panx1, the host cells did not liberate ATP upon stimulation, making it unclear whether Panx1 is involved in taste-related ATP secretion. This issue was addressed using mice with genetic ablation of the Panx1 gene. The ATP-biosensor assay revealed that, in taste cells devoid of Panx1, ATP secretion was robust and apparently unchanged compared with the control. Our data suggest that Panx1 alone forms a channel that has insufficient permeability to ATP. Perhaps, a distinct subunit and/or a regulatory circuit that is absent in taste cells is required to enable a high ATP-permeability mode of a native Panx1-based channel.

Fig. 1.

Representative carbenoxolone-sensitive WC currents recorded from cells of different lines transfected with the pIRES2-EGFP-Panx1 plasmid, including neuroblastoma SK-N-SH (A), CHO (B), and HEK-293 (C) cells. (A–C) Cells were held at −50 mV and polarized by 100-ms (SK-N-SH) or 50-ms (CHO and HEK-293) voltage pulses between −70 and 80 mV [upper panel in C]. Bottom panels: whole-cell (WC) currents were recorded in the presence of 25 µM carbenoxolone (CBX). In all cases, cells were perfused with the solution (mM): 140 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES-NaOH; the patch pipette contains (mM): 100 CsCl, 0.5 MgCl₂, 10 BAPTA-40 CsOH, 10 mM HEPES-CsOH. (D–F) I–V curves of steady-state currents shown in A–C, respectively. The current values for generating I–V curves were measured at the moments indicated by the symbols above the current traces. (G–I) I–V curves of Panx1 currents with different anions in the bath, as indicated in G. In all cases, 140 mM NaCl+2 mM KCl in the bath was substituted for 140 mM Na-gluconate+2 mM K-gluconate or 140 mM NaMeSO₃+2 mM KMeSO₃.

Fig. 2.

Panx1-currents in HEK-293 cells recorded with different anions in the bath. (A–C) Representative I–V curves illustrating shifts of Panx1 current reversal potentials upon replacement of extracellular Cl⁻ with large anions. Here, 140 mM NaCl+2 mM KCl in the bath was substituted for 140 mM Na-gluconate+2 mM K-gluconate, 140 mM Na-glutamate+2 mM K-glutamate, or 140 mM Na-aspartate+2 mM K-aspartate. The Panx1 current was calculated as the difference between whole-cell (WC) currents with and without 25 µM CBX. (D) Panx1 current I–V curves generated by the voltage ramp from −80 mV to 80 mV at different Cl⁻ concentrations in the bath ([Cl⁻]_out), obtained by the replacement of chloride with HEPES so that [Cl⁻]_out+[HEPES]_out = 146 mM. (E) Shift of Panx1 current reversal potential as a function of bath Cl⁻ concentration. The data are presented as a mean ± S.E. (n = 3–7). The solid line corresponds to the equation ΔV_r = −58.6 log([Cl]_out/146). In A–D, cells were dialyzed as in Fig. 1.

Fig. 3.

Single Panx1 channel activity. (A,C) Single-channel current fluctuations observed at different voltages in control (A), and with 20 µM carbenoxolone (CBX) in the bath (C). An inside-out patch was excised from the plasma membrane of a Panx-positive HEK-293 cell. The patch pipette was filled with the solution (mM): 140 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES-NaOH; the bath contains (mM): 140 CsCl, 0.5 MgCl₂, 1 EGTA, 10 HEPES-CsOH. (B,D) Amplitude histograms of patch current recorded at 70 mV in the absence (A) and in the presence of 20 µM CBX in the bath (C) illustrate that two individual channels were active in the patch. The histograms were fitted with the expression (smooth thick lines): with N₁ = 24.5, i₁ = 0.29 pA, σ₁ = 0.61 pA; N₂ = 207, i₂ = 3.31 pA, σ₂ = 0.62 pA; N₃ = 355, i₃ = 6.09 pA, σ₃ = 0.69 pA in B and N₁ = 818, i₁ = 0.14 pA, σ₁ = 0.34 pA; N₂ = 180, i₂ = 3.27 pA, σ₂ = 0.51; N₃ = 20.4, i₃ = 6.42 pA, σ₃ = 0.79 pA in C. Given that i₃- i₂ = 2.78 pA and i₂ -i₁ = 3.02 pA in B and i₃- i₂ = 3.25 pA and i₂ -i₁ = 3.13 pA in D. It appears that 20 µM CBX did not affect magnitude of the single Panx1 channel current in D.

Fig. 4.

Voltage dependence of the Panx1 current. (A) Single Panx1 channel current versus membrane voltage. The data are presented as a mean ± S.E. (n = 6–11). The solid curve represents a combination of two liner dependencies with the slopes of 15 pS and 74 pS at negative and positive voltages, respectively. (B) Dependencies of single Panx1 channel current (•) and integral Panx1 current (▿) on membrane voltage. The data are presented as a mean ± S.E. and are normalized on the value of the particular current at 80 mV. (C) Voltage dependence of the factor NP_o = I/I, calculated using the data in (B). The solid curve corresponds to Eqn 1.

Fig. 5.

Assay of ATP release in Panx1-positive HEK-293 cells. (A) Representative concurrent recordings of a whole-cell (WC) current from a HEK-293 cell (middle panel) and fluorescence from a nearby Fura-4-loaded COS-1 cell (bottom panel). The upper panel illustrates the time course of holding potential and moments of bath application of 25 µM CBX and 100 nM ATP. (B) Depolarization (upper panel) of a type II cell from a wild-type (WT) CV papilla elicited a large outward current (middle panel), followed by stimulation of the ATP-biosensor due to ATP liberation (bottom panel). The perforated patch approach was employed. In (A) and (B), cells were perfused with the solution (mM): 140 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES-NaOH. In (A), the patch pipette contains (mM): 100 CsCl, 2 MgATP, 10 BAPTA-40 CsOH, 10 mM HEPES-CsOH. In (B), the patch pipette contains (mM): 140 CsCl, 0.5 MgCl₂, 1 EGTA, 0.1 EDTA, 10 HEPES-CsOH, 400 µg/ml Amphotericin B.

Fig. 6.

Assay of ATP release in Panx1 knockout taste cells. (A,D) Taste buds from a WT CV papilla (A) and from Panx^−/− CV papilla (D) that were fixed by the patch pipette, with nearby Fluo-4 loaded COS-1 cells serving as ATP-sensors. (B,E) ATP-sensor responses on stimulation of the taste bud (A,D) by 70 mM KCl, or by the taste mixture of 100 µm cycloheximide, 100 µm neotam, and 300 µm SC45647. (C,F) Summary of experiments with wild-type (WT) taste buds (n = 27) and Panx^−/− taste buds (n = 14) assayed as in A,D, and stimulated by KCl (70 and/or 100 mM) and by the taste mixture. The data are presented as a mean ± s.d. For both WT and Panx^−/− taste buds, ATP, which was liberated on stimulation with 100 mM KCl, 70 mM KCl, or the taste mixture, elicited statistically different (P<0.05, Student’s t-test) responses of the ATP-biosensor (C,F). Meanwhile, ATP release from WT on the particular stimulus was statistically indistinguishable (P>0.05) from that exhibited by Panx^−/− taste buds (compare respective bars in C and F). (G) Left panel, representative whole-cell (WC) currents indicating that an assayed taste cell is of type II. The cell isolated from a CV papilla of Panx1-null mice was polarized by 100-ms voltage pulses of between −100 and 50 mV. Right panel: concurrent recordings of WC current (middle trace) from the same taste cell (G) and Fluo-4 fluorescence in a nearby COS-1 cell (bottom trace). Upper trace illustrates the time course of holding potential. The recording conditions were as in Fig. 5B. (H) Averaged responses of the ATP-biosensor on depolarization of nearby taste cells via the patch pipette or by 100 mM KCl. The first two bars summarize experiments with individual WT taste cells, and the last two represent experiments with Panx^−/− taste cells. The particular stimulation – electrical or by KCl – of individual Panx^−/− taste cells elicited ATP-sensor responses that were statistically indistinguishable (P>0.05) from those obtained in experiments with WT taste cells. In all cases, cells were isolated from CV papillae.