Metabolomic screening identifies LPC-16:0 as a pannexin agonist.

(A) Schematic of screening process. Organic extracts of mouse liver tissues were subjected to polarity-based fractionation and assessed for their ability to stimulate currents in HEK cells expressing Panx1+GS or Panx2 using whole-cell patch-clamp. Active fractions were analyzed by liquid chromatography–mass spectrometry. (B) LC-MS traces for fractions #5-8 from the second round of Panx1+GS activity-guided fractionation. (C) Chemical structure of LPC-16:0. (D) and (E) Representative whole-cell patch-clamp traces (D) and quantification of peak current densities (E) triggered by LPC-16:0. Panx1 (wildtype) was expressed in GnTI- cells while the other constructs were expressed in HEK cells. Voltage-clamp recordings were performed at -60 mV. Blue bars indicate application of LPC-16:0 (7 μM), and orange bars indicate application of carbenoxolone (50 μM). n=5-20. One-way ANOVA followed by Dunnett’s test was used to assess statistical significance. (F) ATP release induced by application of LPC-16:0 (10 μM) to Panx1-expressing GnTI- (left) and Panx2-expressing HEK (right) cells. Data are expressed as percent of total ATP released upon membrane solubilization. n=6-8. P values were calculated using unpaired t-test with unequal variances. An asterisk denotes p<0.01 for all tests. Error bars represent s.e.m.

Cellular mVenus-quench assay reveals a family of lysophospholipids as pannexin agonists.

(A) Cartoon illustrating the principle of the mVenus quench assay. (B)-(E) Representative traces ((B) for Panx1 and (D) for Panx2) and quantification of initial mVenus quenching rates ((C) for Panx1 and (E) for Panx2). LPC-16:0 (30 μM) was applied with or without CBX (50 μM), and the maximum mVenus quenching was measured after cell solubilization with 1% Triton-X100. n=8-14. P values were calculated using unpaired t-test with unequal variances. (F) and (G) Initial mVenus quenching rates of Panx1 expressed in GnTI- cells (F) and Panx2 expressed in HEK cells (G). Pannexin activation was induced by 60 μM of saturated sn-1 LPCs (LPC-12:0-20:0), sn-2 LPC (LPC2-16:0), a monounsaturated sn-1 (LPC-18:1), or other sn-1 lysophospholipids with different headgroups (LPA-16:0, LPI-16:0, LPE-16:0, and SPC-18:1). n=4-14 for all conditions. P values were calculated using one-way ANOVA followed by Dunnett’s t-test. Asterisks denote p<0.01. Error bars represent s.e.m.

Functional reconstitution of Panx1 confirms direct activation by LPC16:0.

(A) Schematic representation of YO-PRO-1 uptake assay. (B) A schematic representation of the full length Panx1 construct used for functional reconstitution. Gray area: plasma membrane; Ex: extracellular; In: intracellular. (C) Relative YO-PRO-1 fluorescence triggered by LPC16:0 (100 μM) with or without CBX (50 μM). Asterisks indicate p< 0.01 using a unpaired t-test. n=6-15. (D) Dose-response profile of Panx1 treated with LPC-16:0. Dose responses were fitted with the Hill equation, and the EC50 values are indicated. n=10-13. Error bars represent s.e.m.

Pannexins mediate lysophospholipid signaling.

(A) Schematic illustrating lysophospholipid signaling. (B)-(E) Pannexin activities triggered by extracellularly applied stimuli. Normalized initial mVenus quenching rates are shown for PLA1 (B), sPLA2 (C), and major metabolic products of PLA2 (D), mastoparan with or without PLA2 inhibitors (QCT and CPZ), a Src kinase inhibitor (PP2), or a caspase inhibitor (ZVAD)(E). n=4-14. (F) Panx1-dependent mVenus quenching induced by synovial fluids obtained from canine patients with mild (-) or moderate/severe (+/++) pain. The activity of each fraction was normalized to the effect of LPC-16:0 (30 μM). Each point represents a different patient. n=10-12. P values were calculated using unpaired Student’s t-test with unequal variances ((B) and (C)) or using one-way ANOVA, followed by Dunnett’s t-test ((D) and (E)). Asterisks indicate p<0.01. Error bars represent s.e.m.

LPC induces a conformational change in the N-terminal domain.

(A) Molecular docking of LPC16:0 in the human Panx1 structure (PDB: 7F8J). Two protomers are used for the docking and superposition of the 10 most energetically favorable binding modes of LPC16:0 are shown as stick representations. (B) Close-up view of the predicted LPC16:0 binding pocket. Residues whose Asn mutations abolish LPC-induced currents are shown in red and those that preserve such currents are shown in blue. (C) Ratio of LPC-induced currents (7 μM) over voltage-induced currents (+110 mV) for the indicated double Asn mutants. Panx1+GS background was used for the whole-cell recordings from HEK cells. n=5-11. P values were calculated using one-way ANOVA followed by Dunnett’s t-test. Asterisks denote p<0.01. Error bars represent s.e.m. (D) EM maps of Panx1 in its resting state (frPanx1-ΔLC (3.0 Å)), ‘primed’ state (frPanx1-ΔLC+GS (3.4 Å)), or activated state (frPanx1-ΔLC+GS+LPC (3.2 Å)). Each map represents only four of the seven protomers, for clarity. Map densities corresponding to the NTD are highlighted in red for the resting and primed states, or in blue for the activated state.