The serine protease, furin, is involved in the activation of a number of proteins most notably epithelial sodium channels (ENaC). The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. UT-A1's amino acid sequence has a consensus furin cleavage site (RSKR) in the N-terminal region. Despite the putative cleavage site, we find that UT-A1, either from the cytosolic or cell surface pool, is not cleaved by furin in CHO cells. This result was further confirmed by an inability of furin to cleave in vitro an 35S-labeled UT-A1 or the 126 N-terminal UT-A1 fragment. Functionally, mutation of the furin site (R78A, R81A) does not affect UT-A1 urea transport activity. However, deletion of the 81-aa N-terminal portion does not affect UT-A1 cell surface trafficking, but seriously impair UT-A1 urea transport activity. Our results indicate that UT-A1 maturation and activation does not require furin-dependent cleavage. The N-terminal 81-aa fragment is required for proper UT-A1 urea transport activity, but its effect is not through changing UT-A1 membrane trafficking.
The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca2+]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca2+]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca2+]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca2+]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca2+]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca2+]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca2+]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca2+]i, creating [Ca2+]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca2+]i uptake destroyed the polarized response of ENaC to [Ca2+]i. Overall, our data suggest that ENaC is regulated by [Ca2+]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca2+]i sequestration.
Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.
Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca 2+ -permeable channel, lovastatin decreased, but Cho increased, intracellular Ca 2+ also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca 2+ .
The UT-A1 urea transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature N-acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation by tunicamycin or elimination of the glycosylation sites by mutation significantly reduced UT-A1 activity in oocytes. Taken together, our study reveals a new role of N-linked glycosylation in regulating UT-A1 activity by promoting UT-A1 trafficking into membrane lipid raft subdomains.—Chen, G., Howe, A. G., Xu, G., Fröhlich, O., Klein, J. D., Sands, J. M. Mature N-linked glycans facilitate UT-A1 urea transporter lipid raft compartmentalization.