Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

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.