Pharmacological characterization of pannexin-1 currents expressed in mammalian cells

Abstract

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone > disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) approximately disodium 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate approximately 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X(7) receptor (P2X(7)R), DIDS was found to act as a P2X(7)R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X(7)R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X(7)R activation.

Fig. 1.

Characterization of human Panx1 variants. A, multiple alignment of the different human Panx1 variants (Panx1a AAK91713, Panx1b NP_056183, Panx1bv FM201789) with mouse Panx1 (NP_062355) corresponding to the residues 372 to 407 of the intracellular C terminus in human Panx1a. B, expression profile of Panx1 variants by reverse transcriptase-PCR in different human cell lines and in the brain. Note the lack of Panx1a expression. C, detection of Panx1b and Panx1bv by PCR in the genomic DNA of HeLa and THP-1 cells. Expression vectors for the different Panx1 variants were used as a positive control to confirm the specific amplification by the different oligonucleotide combinations (B and C). D, cellular localization of the different Panx1-ee-tagged variants expressed in HEK293 by confocal microscopy. Note similar membrane localization for all variants.

Fig. 2.

Functional expression of mouse and human Panx1. A, currents in response to voltage steps from -120 to 80 mV and voltage ramps from -120 to 80 mV recorded from HEK293 cells expressing mPanx1. Lanthanum (2 mM) blocks the inward but not the outward current, whereas CBX (30 μM) blocks only the outward current. We have defined the Panx1 current as the CBX-sensitive component. B, CBX inhibition of human Panx1 currents; C, rapid onset of CBX inhibition. D, frequency of expression of Panx1 currents from cells transfected with the indicated constructs; in all transfections, equivalent percentage of cells (> 80%) expressed Panx1 protein as determined by immunohistochemistry (as per Supplemental Fig. 1). E, mean current at 70 mV recorded from cells expressing Panx1 currents; there were no significant differences among the human variants or between mouse and human Panx1 currents.

Fig. 3.

Panx1 currents are not modulated by calcium. A, mPanx1 currents recorded with low internal calcium (<1 nM), high internal calcium (720 nM) with 2 mM external calcium, or low internal calcium and zero external calcium. B, activation of Trp-like currents in response to U73122 (10 μM, blue traces recorded at 1-min intervals). C, currents in absence (black trace) or presence of 2 mM lanthanum (blue traces at 1-min intervals), which show no change in CBX-sensitive Panx1 current over this time course. D, currents in U73122 + lanthanum (blue traces at 1-min intervals); Panx1 currents are now inhibited. E, PLC-inactive analog, U73343 (10 μM), also inhibited Panx1 currents.

Fig. 4.

Kinetics of calcium-independent inhibition of Panx1 currents by U73122 and U73343. Summary of all experiments as illustrated in Fig. 3. A, inhibition by U73122 as a function of time in different internal/external calcium concentrations; there were no significant differences. B, kinetics of inhibition by U73122 and U73343; inhibition by U73343 was slower than U73122, but the same maximal inhibition occurred with either compound.

Fig. 5.

Inhibition of Panx1 currents by some chloride channel/anion transporter inhibitors. A, two examples of concentration-dependent inhibition of mPanx1 currents by NPPB (left) and SITS (right); note NPPB inhibited inward and outward current; lanthanum (2 mM) present throughout. B, concentration-inhibition curves for inhibitory compounds; each point is mean ± S.E.M. of five to 12 points. C, histogram, mean currents in the presence of 100 μM of each of the compounds, except for CBX, which was 50 μM.

Fig. 6.

Extracellular nucleoside triphosphates inhibit Panx1 currents. Voltage steps (A) and ramps (B) in control and presence of ATP as indicated. C, concentration inhibition curves for ATP, UTP, and GTP inhibition of mPanx1 as indicated; n = 5 to 14 for each point. D, currents recorded with high intracellular ATP (20 mM); no differences in peak current or inhibition by extracellular ATP or CBX were observed. E, Panx1 currents in presence of maximal concentrations of nucleotides, nucleosides, and phosphates as indicated.

Fig. 7.

Actions of Panx1 inhibitors on ATP-evoked currents in cells coexpressing Panx1 and P2X₇ receptor. A, examples of ATP-evoked currents in the presence and absence of CBX, NPPB, and MFQ as indicated; currents were slightly increased or unaltered. B, example of ATP-evoked currents in the absence and presence of DIDS. C, summary of all experiments as illustrated in A and B; results for NPPB and MFQ at each of the three P2X₇Rs are pooled because there was minimal or no effect, whereas inhibition by DIDS is shown for each species, n = 5 to 18 for each point. DIDS IC₅₀ values were 90 ± 4, 130 ± 12, and 195 ± 21 μM for rat, mouse, and human P2X₇R, respectively.