Stretch-Induced Activation of Pannexin 1 Channels Can Be Prevented by PKA-Dependent Phosphorylation

Abstract

Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity—measured by changes in DAPI uptake—of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water–lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

“…S4), which presumably prevents dephosphorylation of protein kinase A (PKA)-phosphorylated residues present in both rPanx1 and hPanx1. PKA-dependent phosphorylation was recently shown to prevent activation of the rPanx1 channel (40).…”

“…This value shows that in the rPanx1S394Phos protein those residues are more available to interact with DAPI, favoring its translocation from the extra-to the intracellular side. Additionally, we built a third system, introducing phosphate groups in T302 and S328 previously shown to be phosphorylated by PKA and maintain Panx1 channels insensitive to MS (40), in addition to S394. We obtained electrostatic potential maps of the three systems (rPanx1, rPanx1S394Phos, and rPanx1 S394Phos/ T302Phos/S328Phos), and in the case of the triple phospho form of Panx1 the section of lateral tunnels became much more electronegative compared to the rPanx1 or rPanx1S394Phos (Fig.…”

Pannexin Channel Regulation of Cell Migration: Focus on Immune Cells

“…In addition, PANX1 channels are somehow mechanosensitive, although in a lesser extent in comparison to other channels such as Piezo1 and Piezo2 ( 145 ). Still PANX1 could quickly react to changes in the membrane tension ( 96 , 100 , 146 ), providing a fast feedback mechanism in which the opening of the channel can be controlled by the mechanical cues of the microenvironment that surrounds the migrating cell. This could be sustained in time by the activation of enzymes that directly modify the opening of the channel, such as Ca 2+ -sensitive CaMKII ( 72 ), or by a positive feedback with P2 receptors ( 147 , 148 ) ( Figures 2 , 3 ).…”

“…Indeed, mechanical stretch activates ERK through EGF receptor activation, triggering cell contraction ( 95 ). PANX1 channel opening is also affected by mechanical stress, although some evidence suggests that this occurs in an indirect fashion ( 96 ). However, regardless of the pathways activated PANX channel activity is affected by mechanotransduction and therefore these channels might contribute during cell migration and deformation of extracellular matrix, as it occurs during mesenchymal migration.…”

Mast Cell and Astrocyte Hemichannels and Their Role in Alzheimer’s Disease, ALS, and Harmful Stress Conditions

“…The activity of this channel can be enhanced by an increase of intracellular Ca 2+ concentration, alkaline pH, high extracellular potassium ion concentration, and extracellular ATP [ 49 , 57 , 59 , 60 , 61 , 62 ]. In contrast, the activity of this channel is reduced via phosphorylation by protein kinase A (PKA) [ 63 ]. Upon prolonged opening of this channel, a high amount of ATP (>1 mM) can be released, which can promote neuronal cytotoxicity through the activation of P2X receptors.…”