P190 Regulates The Membrane Localization Of Na~+/K~+-ATPase In Brain Microvascular Endothelial Cells By Interaction With ATP1B3

Purpose:The blood-brain barrier?BBB?is a physiological barrier in the brain that regulates the movement of ions and molecules into and out of the brain and maintains the stability of the central nervous system.BBB is mainly composed of brain microvascular endothelial cells?BMECs?,basement membrane,pseudopods of astrocytes,and pericytes.BMECs are the main constituent cells that make up the blood-brain barrier,and are different from other tissues'endothelial cells.BMECs have tight junctions,adhesive junctions,and low levels of endocytic exocytosis,which limit the passage of small molecules in the blood into the brain through trans-cell and intercellular transport through the BBB.The BMECs membrane surface also has a variety of substrate-specific transport systems that are capable of transporting ions,cellular metabolites and specific small molecules.For example,a sodium-potassium pump?also known as Na⁺/K⁺-ATPase?localized to the surface of the BMECs regulates sodium ions in the cells into the interstitial fluid while pumping potassium ions into the cells.P190,a 190 kD transmembrane protein,is originally found to exist in node of Ranvier of myelinated nerve fibers.For the first time in our laboratory,P190 was expressed in the vascular surface of human brain microvascular endothelial cells?HBMEC?,participation as a receptor in bacterial invasion and passage of BBB leads to neonatal bacterial meningitis?Nature communications,2018?.In order to investigate the physiological significance of expression P190 in BMECs,we used C-terminal domain of P190?80 amino acid?as bait protein to screen probable proteins interacted with P190 by yeast two-hybrid technique,and Na⁺/K⁺-ATPase?₃ subunit?ATP1B3?was found to interact with P190.Na⁺/K⁺-ATPase,also known as sodium potassium pump,maintains the steady state of intracellular low sodium and high potassium ion concentration by consuming ATP while transporting two potassium ions into the cell and transporting three sodium ions to the outside of the cell.The Na⁺/K⁺-ATPase consists mainly of a catalytic?subunit and a regulatory?subunit.The?subunit can help the?subunit transport to localize to the plasma membrane and exercise the enzyme activity of the Na⁺/K⁺-ATPase transport ion.This study will clarify the mechanism of interaction between P190 and ATP1B3 and the possible physiological implications.Methods:ATP1B3 protein was translated in vitro,and whether there was interaction between P190 and ATP1B3 was detected by GST-pull down technology.Immunofluorescence was used to detect the positional distribution of P190 and ATP1B3 in HBMEC.Whether endogenous P190 and ATP1B3 protein in HBMECs interacted with each other was detected by CO-IP technology.ATP1B3 glycosylation was detected by western blot.In HBMEC,P190 protein was knocked down by transient interference with P190 or shRNA.The expression of ATP1B3 and ATP1A1was detected by immunofluorescence and Western blot.In the stable and low expression of P190 HBMEC,the Rescue experiment restored the expression of P190protein,and the expression of ATP1B3 and ATP1A1 was detected by Western blot.Two-photon laser confocal microscopy was used to record the changes of K ion fluorescence signal in HBMEC with low expression of P190 protein.The functional activity of Na⁺/K⁺-ATPase was detected by an activity detection kit.The in vitro BBB model was simulated by transwell cultured with stably low expression P190 HBMEC to detect the changes in the ability of BBB to transport glutamate.Result:The GST-pull down assay demonstrated that P190 interacts with the Na⁺/K⁺-ATPase?₃ subunit?ATP1B3?and does not interact with the?₁ subunit.Cellular immunofluorescence showed that P190 and ATP1B3 colocalized in the endoplasmic reticulum region.ATP1B3 in HBMEC is a glycoprotein with a modified form of different glycosylation.Immunoprecipitation experiments showed that P190protein interacted with unglycosylated ATP1B3.After stable and low expression of P190 protein in HBMEC,the level of glycosylation of ATP1B3 was decreased and the localization of plasma membrane was also reduced.The?subunit?ATP1A1?further affected by the?₃ subunit also undergoes down-regulation of protein expression and decreased cell membrane localization;Stable and low expression of P190 protein can restore ATP1B3 and ATP1A1 expression and localization on the plasma membrane after rescue P190 protein.Elisa results show that interference with P190 in HBMEC can reduce Na⁺/K⁺-ATPase activity.Detection of low expression of P190 protein in HBMEC by two-photon laser confocal microscopy,the relative velocity and total amount of potassium ion transported by Na+/K+-ATPase were significantly reduced.Transwell culture stably interferes with P190 cells and simulates an in vitro BBB model,Compared with normal cells,the cell's ability to transport glutamate from the basal surface to the luminal surface is reduced.Conclusion:P190 protein interacts with unglycosylated ATP1B3 in brain microvascular endothelial cells,Its physiological significance is to promote glycosylation modification of ATP1B3 in brain microvascular endothelial cells?ie,maturation of ATP1B3?.P190-mediated maturation of ATP1B3 promotes assembly of the Na⁺/K⁺-ATPase?₁ subunit and cell membrane localization of Na⁺/K⁺-ATPase.At the same time,P190 regulates the ion transport activity of Na⁺/K⁺-ATPase.It is also beneficial for brain microvascular endothelial cells to transfer excess glutamate into the blood from the brain.