Pannexin channels and ischaemia

Abstract

An ischaemic stroke occurs during loss of blood flow in the brain from the occlusion of a blood vessel. The ischaemia itself comprises a complex array of insults, including oxygen and glucose deprivation (OGD), glutamate excitotoxicity, acidification/hypercapnia, and loss of sheer forces. A substantial amount of knowledge has accumulated that define the excitotoxic cascade downstream of N-methyl-d-aspartate receptors (NMDARs). While the NMDAR can influence numerous downstream elements, one critical target during ischaemia is the ion channel, pannexin-1 (Panx1). The C-terminal region of Panx1 appears critical for its regulation under a host of physiological and pathological stimuli. We have shown using hippocampal brain slices that Panx1 is activated by NMDARs through Src family kinases. However, it is not yet certain if this involves direct phosphorylation of Panx1 or an allosteric interaction between the channel's C-terminal tail and Src. Interestingly, Panx1 opening during ischaemia and NMDAR over-activation is antagonized by an interfering peptide that comprises amino acids 305-318 of Panx1. Thus, targeting the activation of Panx1 by NMDARs and Src kinases is an attractive mechanism to reduce anoxic depolarizations and neuronal death.

Figure 1. Schematic representation of the anoxic depolarization in relation to ATP concentration and glutamate release

Ischaemia causes depletion of intracellular ATP, which is thought to be important for inhibition of the Na⁺–K⁺ pump. Na⁺–K⁺ pump inhibition probably initiates membrane depolarization, as depicted in the lower cartoon of E_m. The putative timing of activation of NMDARs following a transient increase in glutamate release is indicated. NMDARs probably contribute to depolarization, but neuronal death is proposed to occur following activation of downstream cation channels such as pannexin-1 and TRPM7.

Figure 2. Schematic depiction of Panx1 and a model for the role of NMDA receptors and pannexin-1 (Panx1) in ischaemia-induced neuronal death

A, schematic representation of a single Panx1 peptide showing the location of known posttranslational modifications that alter function. The black bars show the known sites of action of three antagonists that block the anoxic depolarization. Panx_305–318 and ¹⁰panx are small peptides and anti-Panx1 is a polyclonal antibody. Green arrows represent putative S-nitrosylation sites, red arrows putative phosphorylation (by Src kinases), blue arrow the caspase 3/7 cleavage site and the black arrow is the known site for N-linked glycosylation. B, functional Panx1 channels are hexameric. Interruption of blood flow (ischaemia) induces glutamate and glycine (gly)/d-serine (D-ser) release. This causes over-activation of the NMDAR, which is a putative pathway for excitotoxic calcium influx. Additionally, NMDARs can activate Panx1 via Src family kinases, leading to exasperated calcium influx. Calcium buffering by mitochondria can lead to permeability transition and cell death. ND2 is NADH dehydrogenase 2. Theoretically, any strategy to disrupt this putative complex could be effective at reducing anoxic depolarizations.