Abstract
Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.
Highlights
Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates ␣1-adrenergic receptor (␣1-AR) vasoconstriction and blood pressure homeostasis
The present study demonstrates the direct phosphorylation of a critical amino acid residue (Tyr198) on the PANX1 intracellular loop by SRC kinase in vascular smooth muscle cells (VSMCs) and the sufficiency of SRC family kinase (SFK) activity to modulate PANX1 channel function and adrenergic mediated vasoconstriction
We find that phosphorylation of the PANX1 Tyr198 residue by SFK activity is constitutive in nature and likely supports the initiation of purinergic signaling cascades at the plasma membrane
Summary
SRC family kinases regulate phenylephrine-induced pannexin 1 channel function independent of Ca2؉. Due to the significant inhibitory effects of SFK-modulating agents on adrenergic mediated vasoconstriction, ATP release, and PANX1 Tyr198 phosphorylation in VSMCs, we set out to address whether SRC kinase modulates PANX1 Tyr198 phosphorylation, as was observed in other cell types [18, 19]. Using co-immunoprecipitation from hCoSMCs, we observed an association of PANX1 with SRC kinase that was independent of adrenergic stimulation (Fig. 5C) Based on these results, we propose that SRC kinase associates with PANX1 at the plasma membrane and phosphorylates PANX1 Tyr198 to support adrenergic mediated channel activation. We treated cells with PP2 to inhibit SFK activity and found a loss of pPANX1Y198 signal at the plasma membrane (Fig. 6B) From these observations, we reasoned that the phosphorylation state at Tyr198 was a specific marker for the pool of activatable plasma membrane–associated PANX1. Phosphorylation of PANX1 Tyr198 is likely an important marker for plasma membrane PANX1 and could be utilized to identify the level of activatable PANX1 channels that associates with vascular pathologies found in sympathetic nerve-mediated hyperstimulation commonly observed in hypertension
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