Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.
The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand–channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.