Structural and functional analysis of human pannexin 2 channel

Abstract

The pannexin 2 channel (PANX2) participates in multiple physiological processes including skin homeostasis, neuronal development, and ischemia-induced brain injury. However, the molecular basis of PANX2 channel function remains largely unknown. Here, we present a cryo-electron microscopy structure of human PANX2, which reveals pore properties contrasting with those of the intensely studied paralog PANX1. The extracellular selectivity filter, defined by a ring of basic residues, more closely resembles that of the distantly related volume-regulated anion channel (VRAC) LRRC8A, rather than PANX1. Furthermore, we show that PANX2 displays a similar anion permeability sequence as VRAC, and that PANX2 channel activity is inhibited by a commonly used VRAC inhibitor, DCPIB. Thus, the shared channel properties between PANX2 and VRAC may complicate dissection of their cellular functions through pharmacological manipulation. Collectively, our structural and functional analysis provides a framework for development of PANX2-specific reagents that are needed for better understanding of channel physiology and pathophysiology.

Fig. 1. Structure of human PANX2.

a Domain topology of human PANX2. The engineered protein construct PANX2_EM for structure determination is indicated. b Current–voltage curves for the wild-type full-length human PANX2 and PANX2_EM expressed in HEK293T cells. Currents from untransfected cells were used as the negative control. c Current density for the full-length and PANX2_EM. Data are presented as mean ± SEM. p-value is indicated (unpaired two-tailed t-test, n = 7, 6 independent cells for the full length and PANX2_EM, respectively). d Cryo-EM reconstruction of PANX2_EM with each subunit uniquely colored. The micelle densities are indicated. Lipid-like densities within the membrane region are colored in orange. e, f Structure of human PANX2 in orthogonal views. g Structure of a single subunit with secondary structures indicated. The disulfide bonds between conserved cysteine residues in the extracellular domain are highlighted.

Fig. 2. Fenestration in PANX2 and PANX1.

a Lateral opening between two adjacent PANX2 subunits. Lipid-like densities (in orange) are numbered as lipid 1 to 4. b Lateral opening between two neighboring PANX1 subunits (PDB: 6UZY). Interfacial lipid densities (in orange) are similarly numbered.

Fig. 3. Ion conduction pore of PANX2.

a The pore of PANX2The extracellular constriction formed by a ring of seven arginine amino acids (R89) from E1H is highlighted. Only two opposing subunits are shown in the top panel for clarity. The extracellular and intracellular constrictions, which are defined by R89 and the N-terminal helices, respectively, are highlighted in the bottom panels. b Pore dimension along the conduction path.

Fig. 4. Pore properties of PANX2, PANX1, and LRRC8A channels.

a Superposition of PANX2 (green) and PANX1 (blue) protomers (PDB: 6UZY). b Superposition of the heptameric PANX2 and PANX1 channels. Only two subunits are shown. c Pore profiles of PANX2 and PANX1. d Overlay of heptameric PANX2 (green) and hexameric LRRC8A (gray, PDB: 6NZW) channels based on superposition of a single protomer from each channel. e Pore profiles of PANX2 and LRRC8A. f–h The putative extracellular selectivity filters of PANX2 (f), PANX1 (g), and LRRC8A (h). i–k Cutaway views of the central pores of PANX2 (i), PANX1 (j), and LRRC8A (k) colored by surface electrostatic potential (red, −5 kT/e; white, neutral; blue, +5 kT/e).

Fig. 5. Pharmacology of human PANX2.

a, b Current–voltage relationship of PANX1 (a) and PANX2 (b) in the absence or presence of 0.1 mM CBX. c Current densities of PANX1- and PANX2-mediated whole-cell currents in the absence or presence of 0.1 mM CBX (mean ± SEM, n = 5 independent cells, unpaired two-tailed t-test). d, e Current–voltage relationship of PANX1 (d) and PANX2 (e) in the absence or presence of 75 μM DCPIB. f Current densities of PANX1- and PANX2-mediated whole-cell currents in the absence or presence of 75 μM DCPIB (mean ± SEM, n = 4, 5 independent cells for PANX1 and PANX2, respectively, **** indicates p < 0.0001, unpaired two-tailed t-test).