Revisiting multimodal activation and channel properties of Pannexin 1

Abstract

Pannexin 1 (Panx1) forms plasma membrane ion channels that are widely expressed throughout the body. Panx1 activation results in the release of nucleotides such as adenosine triphosphate and uridine triphosphate. Thus, these channels have been implicated in diverse physiological and pathological functions associated with purinergic signaling, such as apoptotic cell clearance, blood pressure regulation, neuropathic pain, and excitotoxicity. In light of this, substantial attention has been directed to understanding the mechanisms that regulate Panx1 channel expression and activation. Here we review accumulated evidence for the various activation mechanisms described for Panx1 channels and, where possible, the unitary channel properties associated with those forms of activation. We also emphasize current limitations in studying Panx1 channel function and propose potential directions to clarify the exciting and expanding roles of Panx1 channels.

Figure 1.

Panx1 channels of large and linear unitary conductance. (A) Pressure-induced single-channel activity obtained from a Xenopus oocyte heterologously expressing human PANX1 (adapted from Bao et al., 2004). The unitary conductance of pressure/stretch-activated channels was reported elsewhere to be ∼475 pS (Locovei et al., 2006a). (B) High extracellular K⁺-activated single-channel activity obtained by using inside-out patch recording in Xenopus oocytes heterologously expressing human PANX1 (adapted from Bao et al., 2004). Membrane patch was exposed to symmetric 150 mM K⁺. The high-K⁺–activated channel visited multiple subconductance states and displayed a unitary conductance up to ∼475 pS. (C) Intracellular Ca²⁺-induced single-channel activity obtained by using inside-out patch recording in Xenopus oocytes heterologously expressing human PANX1 (adapted from Locovei et al., 2006b). The unitary conductance of Ca²⁺-activated channels was reported to be ∼550 pS (Locovei et al., 2006b). (D) Single-channel activity evoked by caffeine-induced Ca²⁺ release, obtained from rat atrial myocytes infected with adenovirus expressing mouse Panx1 or empty vector (adapted from Kienitz et al., 2011). The caffeine-activated channels showed a unitary conductance of ∼300 pS. (E) O₂/glucose deprivation (OGD)-induced single-channel activity obtained by using cell-attached recording in rat hippocampal neurons (left). Boxed figures are exemplar single-channel opening (top right) and all-point histogram acquired from recordings under control, OGD, and OGD+CBX conditions (bottom right). The OGD-activated channels demonstrated a unitary conductance of ∼530 pS (adapted from Thompson et al., 2006). All figures are reproduced with permission.

Figure 2.

Raising extracellular K does not activate recombinant or native Panx1 channels. (A and B) Whole-cell currents were obtained from HEK293T cells expressing either wild-type PANX1 (A) or C-terminally truncated PANX1 (B) under control conditions (3 mM K⁺), high extracellular K⁺ (83 mM K⁺), and high extracellular K⁺ plus CBX (50 µM); insets show time series of current obtained at 80 mV under the indicated conditions. As previously reported (Chiu et al., 2017), whole-cell voltage-ramp I-Vs (−100 to 80 mV; 0.2 V/s at 0.14 Hz) were obtained at room temperature using borosilicate micropipettes (3∼5 MΩ) filled with internal solution containing (mM) 100 CsMeSO₄, 30 TEACl, 4 NaCl, 1 MgCl₂, 0.5 CaCl₂, 10 HEPES, 10 EGTA, 3 ATP-Mg, and 0.3 GTP-Tris, pH 7.3. Control (3 mM K⁺) bath solution was composed of (mM) 140 NaCl, 3 KCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose, pH 7.3. High-K⁺ solution included (mM) 60 NaCl, 83 KCl, 2 MgCl₂, 2 CaCl₂, and 10 HEPES at pH 7.3; glucose was added to maintain equal osmolarity with the control bath solution (∼300 mOsm). (C) Grouped data (mean ± SEM) shows that CBX-sensitive current from wild-type (n = 5) or CT-truncated PANX1 (n = 7) was unaffected by different extracellular K⁺ concentrations. These results were originally reported in the peer review file from Chiu et al. (2017). (D) Dye uptake measured by flow cytometry shows that viable (7-AAD negative) mouse splenocytes display negligible TO-PRO-3 uptake under control K⁺ conditions (5 mM K⁺; 322 mOsm), hypotonic high-K⁺ (50 mM K⁺, 237 mOsm; same ionic composition as Silverman et al., 2009), or osmolarity-adjusted high-K⁺ (50 mM K⁺ with 87 mM d-mannitol, 327 mOsm). In contrast, caspase-mediated Panx1 activation in UV-irradiated cells yields robust TO-PRO-3 uptake by viable cells. Splenocytes were freshly isolated from C57BL/6 mice, as previously described (Jin et al., 2008) and cultured in growth media (RPMI + 10% FBS); one group of cells was also exposed to UV irradiation (15 × 10⁴ µJ). After 6 h culture at 37°C, cells were washed three times with RPMI, before a 30-min incubation in solutions containing different concentrations of K⁺. TO-PRO-3 (Panx1-permeable) and 7-AAD (Panx1-impermeable) were added ∼10 min before flow cytometry, as previously reported (Poon et al., 2014; Chiu et al., 2017). Note that necrotic cells (7-AAD⁺) were excluded from the analysis to avoid Panx1-independent TO-PRO-3 uptake.

Figure 3.

Panx1 channels of ≤100 pS and outwardly rectifying unitary conductance. (A) Single-channel activity and outwardly rectifying unitary conductance of basally active mouse Panx1 heterologously expressed in HEK293 cells (adapted from Romanov et al., 2012). (B) Single-channel activity of human PANX1 heterologously expressed in HEK293T cells, activated by caspase 3–mediated C-tail cleavage in an inside-out configuration (left). The C-tail cleavage-activated channels demonstrated an outwardly rectifying unitary conductance (top right), whereas the open probability (P_O) remained unchanged across a wide range of membrane voltage (bottom right). Figures were adapted from Chiu et al. (2017). (C) Unitary current amplitudes closely overlay CBX-sensitive whole-cell currents using two-point normalization (left), suggesting that the outwardly rectifying whole-cell current is mainly attributed to the outwardly rectifying unitary conductance. Note that the same data points are not well aligned when normalized to the peak current amplitude at 80 mV (right). All figures are reproduced with permission.

Figure 4.

A general model for Panx1 activation by progressive displacement of autoinhibitory C-tails. (A) Diagram depicts an irreversible activation of Panx1 channels by progressive removal of C-tails. (B) C-tail cleavage-activated human PANX1 channels showed longer open time (top) in contrast to α1D receptor-activated PANX1 channels that demonstrated flickering openings (bottom). (C) A proposed model for activation of Panx1 channels by reversible mechanisms, involving sequential posttranslational modifications or binding of extracellular K⁺ or intracellular Ca²⁺ on Panx1 subunits. All figures are adapted from Chiu et al. (2017) and reproduced with permission.