Role of an Aromatic–Aromatic Interaction in the Assembly and Trafficking of the Zebrafish Panx1a Membrane Channel

Abstract

Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein. Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking.Biomolecules 2020, 10, 272 2 of 15 teleost whole-genome duplication event that occurred between 320 and 350 million years ago, a panx1a ohnologue exists (panx1b) [16]. Panx1a and panx1b show distinct tissue expressions, glycosylation patterns, and electrophysiological gating properties [14]. This study will focus on the panx1a ohnologue.Electrophysiological gating properties and multiple factors regulating the Panx1 channel, as well as complex pharmacology, have been described [17,18]. Panx1 blockers include carbenoxolone, mefloquine, and flufenamic acid, which also act on gap junction proteins. Potassium and glutamate can activate pannexins. Studies across multiple fields demonstrated that Panx1 is a major molecular hub interacting with many signaling pathways. To address the complex life cycle of Panx1, we explored the role of an aromatic-aromatic interaction between amino acids W123 and Y205 in the cytoplasmic loop of panx1a near transmembrane (TM) domains 2 and 3, respectively. Aromatic-aromatic interactions have been previously shown to be important in TM-TM association of membrane proteins [19,20]. The forces of these interactions help strengthen oligomerization and suggest a role in folding and stabilization. Both W123 and Y205 are highly conserved between various membrane channels and gap junction proteins. Mutation analysis of panx1a paired with co-localization and protein interaction studies led to the conclusion that the two aromatic residues are vital for the structural stabilization and interaction of panx1a TM domains before insertion into the cell membrane. Outcomes of this study are relevant to understand how Panx1 proteins mature and traffic to the cell membrane.

Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

“…Recovery kinetics and mobile fraction were comparable to those seen for mobility of other proteins of similar size in a non-junctional membrane (e.g., occludin and Cx30 connexons [ 51 ]). Previous FRAP studies performed at room temperature on GFP-tagged mouse Panx1 expressed in a breast cancer cell line (BICR-MIRk) [ 43 ] and zebrafish Panx1a expressed in N2a cells [ 52 ] revealed diffusion kinetics comparable to those reported here, with recovery of 45–60% at one min after bleaching.…”

Convergent NMDA receptor—Pannexin1 signaling pathways regulate the interaction of CaMKII with Connexin-36

“…Next, we tested the functional coupling of wild-type Cx36 and the R278A mutant using an ethidium bromide uptake and recovery after photobleaching assay 18,21 . Neuroblastoma 2a (Neuro2a) cell pairs expressing Cx36-EGFP or Cx36-EGFP R278A GJPs were selected to quantify the recovery of ethidium bromide fluorescence in the cytoplasm in 0.5-1 µm distance to GJPs after photobleaching one side of the channels (Supplementary Fig.…”

“…FRET analysis. Protein interactions at GJPs were investigated using the acceptor bleach protocol 18,21 . Neuro2a cells were transfected with DsRed-monomer and EGFP/ECFP-tagged expression vectors.…”

Endocytosis of Connexin 36 is Mediated by Interaction with Caveolin-1

“…Cells were fixed with 4% paraformaldehyde and mounted on coverslips. Zeiss LSM 700 confocal microscope was used under a previously established acceptor bleach protocol [ 71 ]. Baseline readings were recorded prior to the acceptor bleach protocol.…”