Abstract
Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia – histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.
Highlights
Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety ofphysiological contexts
We find that HEK293T cells have no detectable endogenous PANX1 currents, assessed using the channel blocker carbenoxolone (CBX)[5,15]; when transfected in those cells, mPanx[1] generates basal whole-cell currents whereas hPANX1 is basally silent (Supplementary Fig. 1a–c). mPanx[1] and hPANX1 are both activated by co-expressed α1D-adrenergic receptor (α1D-AR; Supplementary Fig. 1a–c), and mPanx[1] is activated by multiple additional GαqPCRs (e.g., H1 histamine receptor, metabotropic glutamate receptor mGluR1; Supplementary Fig. 1e, f)
We found that receptor-mediated Panx[1] activation was largely unaffected when cells were recorded with pipettes containing high concentrations of BAPTA to rapidly chelate intracellular calcium, again consistent with the idea that a Ca2+-dependent signaling pathway is dispensable for channel activation (Supplementary Fig. 2a, b)
Summary
Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. Substitution of basallyacetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism. In this work, using molecular and pharmacological tools, we show that Panx[1] activation by the α1D-AR engages Gαq and an unconventional RhoA-mDia-HDAC6 signaling pathway to mediate direct activation of Panx[1] channels by lysine deacetylation. We demonstrate this α1-AR and HDAC6mediated Panx[1] activation in T lymphocytes, suggesting a potential signaling modality for neuro-immune modulation by GPCRs20
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