Abstract:Renal cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Trafficking of NKCC2 to the apical surface regulates NKCC2-mediated NaCl absorption and blood pressure. The molecular mechanisms by which NKCC2 reaches the apical surface and their role in renal function and maintenance of blood pressure are poorly characterized. Here we report that NKCC2 interacts with the vesicle fusion protein VAMP3, and they co-localize at the TAL apical surface. We observed that si… Show more
“…2f). Meanwhile, the vesicle-associated membrane protein 3 (VAMP3), one of the SNARE family related to intracellular vesicle trafficking 34, 35 , was detected in EM and EhCv/siRNA NPs. Similarly, the protein components retained in ChCv/siRNA NPs were consistent with the electrophoresis strips of cell membrane, but no protein signal was detected in Cv/siRNA NPs without membrane decoration.…”
Section: Resultsmentioning
confidence: 99%
“…2e), in which some proteins (such as VAMP3 (Fig. 2f)) plays an important role for membrane binding and fusion 34, 35 . Therefore, it is reasonable to believe that the decoration of EM on siRNA-loaded cationic vehicle is probably a suitable strategy that can be used to effectively improve cellular uptake.Fig.…”
Most cationic vectors are difficult to avoid the fate of small interfering RNA (siRNA) degradation following the endosome-lysosome pathway during siRNA transfection. In this study, the endoplasmic reticulum (ER) membrane isolated from cancer cells was used to fabricate an integrative hybrid nanoplexes (EhCv/siRNA NPs) for improving siRNA transfection. Compared to the undecorated Cv/siEGFR NPs, the ER membrane-decorated EhCv/siRNA NPs exhibits a significantly higher gene silencing effect of siRNA in vitro and a better antitumor activity in nude mice bearing MCF-7 human breast tumor in vivo. Further mechanistic studies demonstrate that functional proteins on the ER membrane plays important roles on improving cellular uptake and altering intracellular trafficking pathway of siRNA. It is worth to believe that the ER membrane decoration on nanoplexes can effectively transport siRNA through the endosome-Golgi-ER pathway to evade lysosomal degradation and enhance the silencing effects of siRNA.
“…2f). Meanwhile, the vesicle-associated membrane protein 3 (VAMP3), one of the SNARE family related to intracellular vesicle trafficking 34, 35 , was detected in EM and EhCv/siRNA NPs. Similarly, the protein components retained in ChCv/siRNA NPs were consistent with the electrophoresis strips of cell membrane, but no protein signal was detected in Cv/siRNA NPs without membrane decoration.…”
Section: Resultsmentioning
confidence: 99%
“…2e), in which some proteins (such as VAMP3 (Fig. 2f)) plays an important role for membrane binding and fusion 34, 35 . Therefore, it is reasonable to believe that the decoration of EM on siRNA-loaded cationic vehicle is probably a suitable strategy that can be used to effectively improve cellular uptake.Fig.…”
Most cationic vectors are difficult to avoid the fate of small interfering RNA (siRNA) degradation following the endosome-lysosome pathway during siRNA transfection. In this study, the endoplasmic reticulum (ER) membrane isolated from cancer cells was used to fabricate an integrative hybrid nanoplexes (EhCv/siRNA NPs) for improving siRNA transfection. Compared to the undecorated Cv/siEGFR NPs, the ER membrane-decorated EhCv/siRNA NPs exhibits a significantly higher gene silencing effect of siRNA in vitro and a better antitumor activity in nude mice bearing MCF-7 human breast tumor in vivo. Further mechanistic studies demonstrate that functional proteins on the ER membrane plays important roles on improving cellular uptake and altering intracellular trafficking pathway of siRNA. It is worth to believe that the ER membrane decoration on nanoplexes can effectively transport siRNA through the endosome-Golgi-ER pathway to evade lysosomal degradation and enhance the silencing effects of siRNA.
“…Previous studies in isolated TALH have provided key insight into the mechanisms of NKCC2 activation by signaling enzymes. In particular, they revealed that NKCC2 could be recruited at the cell surface from subapical vesicles (Ares, 2019; Ares, Caceres, Alvarez‐Leefmans, & Ortiz, 2008; Caceres, Ares, & Ortiz, 2009; Caceres, Mendez, Haque, & Ortiz, 2016; Ortiz, 2006) and that it underwent phosphorylation under such circumstances (Gimenez & Forbush, 2003; Gunaratne et al, 2010). Even if these studies were conducted to understand the bases of NKCC2 regulation by cAMP or cGMP, they still pointed towards a key role for the trafficking machinery in response to carrier phosphorylation.…”
Na +-K +-Cl − cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na + , K + , and Cl − ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na + , Cl − , Ca 2+ , Mg 2+ , and HCO 3 − loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na +-K +-Cl − cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses.
“…New techniques, including whole-exome/genome sequencing, could identify other genes that can cause BS and GS and increase our knowledge of the exact mechanisms of tubular solute handling [3,4,21]. For instance, the recent discovery that vesicle-associated membrane protein 3 (VAMP3) is needed for accurate intracellular trafficking of NKCC2 to the luminal membrane, and that Vamp3 -/mice exhibit a BS phenotype, would make this an interesting candidate gene [51]. Also, mutations in genes involved in the phosphorylation of NKCC2 could decrease its activity.…”
Bartter and Gitelman syndromes are rare inherited tubulopathies characterized by hypokalaemic, hypochloraemic metabolic alkalosis. They are caused by mutations in at least 7 genes involved in the reabsorption of sodium in the thick ascending limb (TAL) of the loop of Henle and/or the distal convoluted tubule (DCT). Different subtypes can be distinguished and various classifications have been proposed based on clinical symptoms and/or the underlying genetic cause. Yet, the clinical phenotype can show remarkable variability, leading to potential divergences between classifications. These problems mostly relate to uncertainties over the role of the basolateral chloride exit channel CLCNKB, expressed in both TAL and DCT and to what degree the closely related paralogue CLCNKA can compensate for the loss of CLCNKB function. Here, we review what is known about the physiology of the transport proteins involved in these disorders. We also review the various proposed classifications and explain why a gene-based classification constitutes a pragmatic solution.
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