A quantized mechanism for activation of pannexin channels

Abstract

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.

Figure 1. C-terminal cleavage by caspase 3 results in a distinctive PANX1 pore structure and maximum unitary conductance of ∼96 pS.

(a) Electron micrograph and class-averaged EM images of negatively stained full-length (from 125 or 282 of 5,970 particles for the putative extracellular or cytoplasmic view, respectively) or caspase-cleaved PANX1 (from 79 or 56 of 6,892 particles); six-fold symmetry was imposed on the indicated images. Schematics show the caspase cleavage site and expected cytoplasmic views of PANX1 hexameric channels, before and after cleavage. Scale bar, 50 nm; class-averaged image: 35.7 × 35.7 nm². (b) Inside–out recordings from HEK293T cells expressing full-length PANX1-FLAG following membrane excision, and after exposure to activated caspase 3 (Casp3) and carbenoxolone (CBX, 50 μM). (c) Steady-state activity of caspase-activated, full-length PANX1-FLAG in an inside–out patch held at different potentials. C, closed state; O1 and O2, open-state amplitude for one and two channels. (d) Averaged single-channel current amplitudes at different patch potentials (± s.e.m., smaller than symbol size) reveal an outward-rectifying unitary conductance: 96.2±2.0 pS from +50 to +80 mV (n=5) and 12.2±0.2 pS from −80 to −50 mV (n=4). (e) Open probability of cleavage-activated PANX1-FLAG is independent of membrane voltage. Data from each patch is represented by a different colour.

Figure 2. PANX1 forms hexameric channels in the plasma membrane.

(a) Schematic of GFP-tagged dimeric and trimeric PANX1 concatemers showing positions of FLAG tag (orange, DYKDDDDK) and TEV protease (TEVp) recognition site (blue, ENLYFQG) in linkers between PANX1 subunits; intact caspase site (red ellipse) is retained in full-length C-terminal domains (red). Linker sequence indicates TEVp cleavage site (blue arrow) and the start methionine (M). Expected hexameric conformations for the holo-channel are also provided. (b) Representative cell-surface biotinylation assay (from n=3) shows GFP-tagged concatemers expressed on the plasma membrane (upper) and in the whole-cell lysate (lower). (c) Single-molecule photobleaching using TIRF microscopy. Histograms show number of spots with different photobleaching steps (n=3 experiments; see insets for photobleaching examples). Note that only two particles from 2(1CT)-GFP showed more than three photobleaching steps and no particles from 3(2CT)-GFP showed more than two photobleaching steps. The lines and symbols overlaid on histograms represent fitted binomial distributions: tetramer (grey square), trimer (red triangle) or dimer (grey circle) for 2(1CT)-GFP; trimer (grey triangle), dimer (red circle) or monomer (grey diamond) for 3(2CT)-GFP. All fits assume ∼65% of GFP fluorescence is available for photobleaching (Methods). (d) Cross-linked (paraformaldehyde (PFA)) dimeric PANX1 concatemers (no GFP tag) are primarily seen at a size expected for hexameric PANX1 (3 × dimer). (e) Whole-cell currents (mean±s.e.m.) were observed from PANX1 concatemers in HEK293T cells only after inter-subunit linkers were cleaved by co-expressed TEVp. Inset: normalized I–V relationships for TEVp-activated, CBX-sensitive currents from monomeric (PANX1(TEV)) and concatemeric PANX1 channels are essentially identical. Number of recorded cells indicated. *P<0.05 using two-way analysis of variance followed by Fisher's least significant difference (LSD) test.

Figure 3. Inside–out patch recordings from HEK293T cells expressing hexameric PANX1 concatemers with different numbers of intact C termini.

(a) Diagram illustrates protocol for inside–out patch recording. (b) Steady-state inside–out patch recordings obtained at +70 mV from HEK293T cells expressing hexameric concatemers with 0 to 6 intact C termini. (c) Electron micrograph and class-averaged images of negatively stained single particles of 6(0CT), before (left, 315 of 13,240 particles were used for class average) and after TEVp cleavage (right, from 38 of 5,883 particles). Images of imposed six-fold symmetry are provided. Scale bar, 50 nm; class-averaged image, 35.7 × 35.7 nm².

Figure 4. Decreasing numbers of PANX1 C termini increase unitary conductance and open probability of PANX1 channel.

(a,b) Single-channel I–V relationships from inside–out patches for PANX1 hexameric concatemers with different numbers of intact C termini recorded after TEVp exposure (a) and after removing remaining C termini with Casp3 (b). Unitary conductance indicated for each hexameric construct. Plotted data are mean±s.e.m., and numbers of recorded patches are provided in parenthesis; for Casp3-cleaved concatemeric hexamers, conductance was ∼95 pS (n=28). (c) Averaged unitary conductance (± s.e.m.) for each of the hexameric concatemers with different numbers of intact C termini (a,b), after TEVp exposure (blue) and from the same patches after Casp3 cleavage (red); data were fitted to a Hill equation, with coefficient -0.3 (blue dash line). The shaded area represents the 95% confidence interval for unitary conductance obtained from all fully cleaved concatemers (that is, after Casp3). (d) Open probability (NP_O; from +80 mV) of PANX1 concatemers with different numbers of C termini (after TEVp) was normalized to that obtained from the same patches after Casp3 cleavage, averaged (±s.e.m.), and fitted by linear regression (blue dash line).

Figure 5. Graded increase in current and ATP/dye permeation via concatemeric PANX1 channels with decreasing numbers of intact C termini.

(a) Whole-cell current density was obtained from HEK293T cells expressing various GFP-tagged hexameric PANX1 concatemers, with (blue) or without (black) TEVp co-expression. Numbers of recorded cells are provided in the parenthesis. Inset: averaged CBX-sensitive I–V relationships, normalized to current density at +80 mV, from cells co-expressing concatemers plus TEVp. Note that 6(6CT) is not depicted because that construct generated no CBX-sensitive current, even with TEVp co-expression. (b) Cell-surface biotinylation from HEK293T cells expressing GFP-tagged hexameric PANX1 concatemers. Representative Western blots (top) and grouped data (bottom, n=4) show a similar ratio of cell-surface expression for all GFP-tagged PANX1 concatemers (normalized to total expression). Loading controls for streptavidin pull-down samples and total cell lysate samples were Na⁺/K⁺ ATPase and α-tubulin. (See Supplementary Fig. 6 for uncropped images). (c) Averaged Trovan-sensitive ATP release from HEK293T cells expressing GFP-tagged hexameric PANX1 concatemers with (blue) or without (black) TEVp co-expression (n=4 experiments; 4 h collection). (d) TO-PRO-3 uptake in Jurkat T cells expressing GFP-tagged hexameric PANX1 concatemers. Representative flow cytometry data shows GFP fluorescence (left) or TO-PRO-3 uptake (right) in cells with or without TEVp co-expression. Bar graph shows grouped data from four independent experiments; mean fluorescence intensity (MFI) of TO-PRO-3 uptake was normalized to the corresponding MFI for GFP within an experiment, and those values were normalized to the value for 6(0CT)+TEVp across independent experiments. Predicted whole-cell current (a), ATP release (c) and TO-PRO-3 uptake (d) based on single-channel properties are overlaid (grey data points). All data are presented as mean±s.e.m.; *P<0.05 using two-way analysis of variance followed by Fisher's LSD test; NS, no statistical significance.

Figure 6. Single-channel activity of PANX1 is gradually increased during apoptosis and upon α1D adrenoceptor-mediated activation.

(a) Cell-attached recording from anti-Fas-treated apoptotic Jurkat T cells after inhibition of caspase activity at 60 min using 25 μM Q-VD-OPh (Q-VD); two CBX-sensitive channels with unitary conductance of either ∼82 pS (O_L1) or ∼58 pS (O_S1) were present in this patch. (b) Unitary conductance for CBX-sensitive PANX1 channels in apoptotic Jurkat T cells, obtained from cells treated with Q-VD at 30, 60 or 120 min of anti-Fas exposure. Data obtained from the same patch are labelled in the same colour. Shaded region represents 95% confidence interval of unitary conductance obtained from C-terminally truncated PANX1 in cell-attached configuration (from d; Supplementary Fig. 1d). (c) Cell-attached recording from HEK293T cells co-expressing α1D adrenoceptors (α1DR) and full-length PANX1-GFP channels obtained at +80 and +60 mV. PANX1 channel activity was not observed before phenylephrine (PE, 20 μM); small conductance channel openings (O_S1, ∼23 pS) observed after ∼4 min of PE exposure showed lower open probability after CBX (see boxed region). A larger conductance channel (O_L1, ∼80 pS) became predominant in the patch after ∼8 min exposure to PE (blue arrows). (d) Outwardly rectifying steady-state single-channel I–V relationships (mean±s.e.m.) from cell-attached recordings of C-terminally truncated PANX1 (red) or α1DR-activated PANX1 (blue), with averaged unitary conductance of 74.7±3.6 pS or 82.7±1.4 pS at positive potentials (n=8 and 4), and 12.8±1.2 pS and 14.9±2.1 pS at negative potentials (n=4 and 3). (e,f) Representative cell-attached recordings at +80 mV from HEK293T cell expressing GFP-tagged, C-terminally truncated PANX1 (e) or α1DR and full-length PANX1 (f), ∼12 min after PE application. (g) Open time distribution from the cell-attached recordings depicted in e,f; mean open time for C-terminally truncated and α1DR-activated PANX1-GFP are ∼7.9 and ∼1.3 ms.

Figure 7. Hypothetical models for PANX1 channel activation.

(a) Schematics illustrate that individual cleavage at the C terminus of PANX1 subunits gradually increase unitary conductance (γ) and open probability (Po). (b) Post-translational modification (yellow star) of α1DR-activated PANX1 subunits gradually increases unitary conductance and open probability by either facilitating displacement of PANX1 C termini (left) or allosterically modulating channel gating via the displaced PANX1 C terminus (right).