Physiological mechanisms for the modulation of pannexin 1 channel activity

Abstract

It is widely recognized that ATP, along with other nucleotides, subserves important intercellular signalling processes. Among various nucleotide release mechanisms, the relatively recently identified pannexin 1 (Panx1) channel is gaining prominence by virtue of its ability to support nucleotide permeation and release in a variety of different tissues. Here, we review recent advances in our understanding of the factors that control Panx1 channel activity. By using electrophysiological and biochemical approaches, diverse mechanisms that dynamically regulate Panx1 channel function have been identified in various settings; these include, among others, activation by caspase-mediated channel cleavage in apoptotic immune cells, by G protein-coupled receptors in vascular smooth muscle, by low oxygen tension in erythrocytes and neurons, by high extracellular K(+) in various cell types and by stretch/strain in airway epithelia. Delineating the distinct mechanisms of Panx1 modulation that prevail in different physiological contexts provides the possibility that these channels, and ATP release, could ultimately be targeted in a context-dependent manner.

Figure 1. C terminal cleavage-mediated Panx1 channel activation using a TEV protease system

A, schematic of a Panx1 (TEV) construct with TEV protease site substituted for the C terminal caspase site. Co-expression of TEV protease results in C terminal cleavage and subsequent Panx1 channel activation leading to YO-PRO-3 dye uptake into the cell. B, when co-transfected with TEVp in HEK293T cells, Panx1(TEV) generated robust whole cell CBX-sensitive currents with I–V properties characteristic of Panx1. C, comparison of Panx1 channel activity assessed by membrane current recording and by dye uptake. HEK293T cells expressing C terminally deleted, constitutively activated (PanxΔC), cleavage activated (Panx1(TEV) + TEVp), and basally inactive Panx1(TEV) were recorded in whole cell voltage clamp mode (left panel), or treated with YO-PRO-3 dye, fixed and fluorescence measured on a plate reader (right panel). YO-PRO-3 dye uptake assay was performed in a 96-well plate format and can reflect differences in Panx1 channel activity as measured by whole cell voltage clamp recordings.

Figure 2. Mechanisms of pannexin1 channel modulation

Schematic outlining direct mechanisms of Panx1 channel modulation including activation via plasma membrane stretch/strain, membrane depolarization, extracellular potassium, low oxygen tension, G protein-coupled receptor, caspase cleavage and ionotropic receptors including P2X. Panx1 can be inhibited directly by high levels of extracellular ATP, and Panx1 channel activity at the level of the plasma membrane can be regulated by trafficking dynamics.