Biology subjects

Bayliss, D. A.

Publications and source records attributed to Bayliss, D. A..

3 recordsLinked to original sources

bioRxiv2026

Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -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. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls 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 mutants 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 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

physiology↗

bioRxiv2024

Novel Pannexin 1 isoform is increased in cancer

Alternative translation initiation (ATI) is a process of increasing protein diversity from one transcript, allowing cells to rapidly respond to signals, which is particularly important in cancer cells. Here, we report potential internal translation start sites exist in PANX1 which have implications in trafficking and channel function. Using mouse (mPANX1) constructs for each internal methionine, we saw that these PANX1 isoforms were N-glycosylated, could traffic to the cell surface and mPANX1-M37 formed functional channels activated by C-terminus cleavage or 1-adrenoceptor stimulation. We also identified a ~25 kDa isoform of mPANX1 (mPANX1-25K) endogenously expressed in mouse melanoma cell lines that could be confirmed with a cognate peptide. mPANX1-25K lacks the mPANX1 N-terminus and most likely corresponds to the M210 internal translation start site since we could not identify any alternative transcripts that would produce this ATI product. When we expressed the human equivalent of M210 in Hs578T PANX1 KO cells with and without wildtype human PANX1, we determined M211 exhibits a predominantly intracellular localization, is N-glycosylated and can interact with full-length human PANX1. Collectively, these findings indicate species specific differences in the abundance of PANX1 ATI isoforms which could act independently or in conjunction with the canonical full-length protein in melanoma.

cell biology↗

bioRxiv2021

A Constitutively Closed Pannexin1 Channel in Lipid Bilayer Nanodiscs Assembles as a Large-Pore Heptamer

Pannexin 1 (Panx1) channels are widely expressed and play important roles in apoptotic cell clearance, inflammation, blood pressure regulation, neurological disorders, opiate withdrawal, and cancer progression and metastasis. We performed (1) physicochemical analysis on a constitutively closed Panx1 channel (designated fPanx1{Delta}C) to examine the entire population of particles to detect multiple oligomeric states and (2) cryoEM in the membrane mimetics amphipol A8-35 and lipid bilayer nanodiscs. Our results reveal that the dominant if not exclusive oligomeric state of fPanx1{Delta}C is a heptamer, in solution and by cryoEM. The Panx1 heptamer provides further structural diversity within the family of large-pore channels, including hexameric LRRC8 (SWELL1) channels and connexin hemichannels, octameric CALHM1 channels and innexin hemichannels, and undecameric CALHM2 channels. Conserved structural themes are a large cytoplasmic vestibule with a diameter that corresponds roughly with the oligomeric state and a 4-helix bundle protomer, albeit with noncanonical helical packing for CALHM1 and CALHM2. In BriefThe 4-helix bundle protomer of a constitutively closed pannexin1 channel assembles as a heptamer in solution and by cryoEM.

biophysics↗