Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate α-adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. During sympathetic-driven hypertension, only Y198F mice showed decreases in blood pressure, with reductions resembling control mice treated with the sympathetic inhibitor clonidine. Pressure myography revealed α-adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A third-order mesenteric arteries; with Y198F responses mimicking control vessels treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing but found a minimal number of differentially expressed genes between phospho-dead mutant and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the α1D-adrenergic receptor and each phospho-dead mutant PANX1 showed phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, S205A and Y308F ion flux was reduced, with ATP release resembling controls. Taken together, these findings demonstrate that physiological outcomes are regulated by distinct PANX1 metabolite versus current conducting properties, which are ultimately controlled by PANX1 phosphorylation.