Abstract
Pannexin 1 (Panx1) is a tetra-spanning, oligomeric, ubiquitously expressed, single membrane protein channel that mediates nucleotides such as ATP release upon activation. Panx1 channels are involved in diverse physiological and pathological processes such as blood pressure regulation, apoptotic cell clearance, inflammation, neurological disorders, and cancer progression and metastasis. Panx1 channels can be activated by various mechanisms including mechanical stress, increased extracellular potassium or intracellular calcium, receptor-mediated signaling pathways, and caspase cleavage of the distal C-terminus. Despite extensive knowledge about the physiology of Panx1 channels, there is much to learn about their structure and molecular basis for channel regulation.
We use negative-stain electron microscopy (EM) and electrophysiology to explore the conformational changes and channel property associated with caspase cleavage-mediated activation. We find that Panx1 channels activated by caspase cleavage display a prominent ‘pore’, voltage-independent gating and outwardly rectifying unitary conductance (<100 pS) at depolarized potentials. Our results support the model that caspase cleavage activates Panx1 channel by removing the pore-associated C-terminal autoinhibitory tails.
We use single-particle electron cryomicroscopy (cryoEM) and electrophysiology to investigate the structure and function of Panx1 channels. We determined the three-dimensional (3D) structure of lipid nanodisc-embedded, C-terminally truncated frog Panx1 at 7 Å resolution. The 3D reconstruction reveals that Panx1 is a heptameric channel, with seven subunits surrounding a central seven-fold symmetric pore axis. The oligomeric state differs from hexameric connexin and octameric innexin hemichannels. A large entrance vestibule resides at the cytoplasmic surface, whereas the extracellular surface displays a narrow pore. The 3D reconstruction clearly resolves the transmembrane (TM) α-helices, which fold as a 4-helix bundle in each subunit. The 4-helix bundle structure of Panx1 recapitulates a similar subunit design with other oligomeric, tetra-spanning membrane protein channels such as connexins (Cxs), innexins (Inxs) and leucine-rich repeat-containing 8 (LRRC8) channels. Rigid body fitting reveals that the monomeric structure of Panx1 resembles LRRC8A channel more than INX-6. Electrophysiological recordings show that the C-terminally truncated Panx1 channels are in a closed conformation, which can be activated by the α1D adrenoceptor-mediated signaling pathway. This result suggests that activation of the α1D adrenoceptor results in phosphorylation of a cytoplasmic site on Panx1 that elicits a transmembrane conformational change to open the extracellular gate. Our work reveals the previously unknown architecture of pannexin channels with respect to the oligomeric state (heptameric), channel and pore dimensions, and the arrangement of the TM helices in each subunit.
