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WNK3 positively regulates epithelial calcium channels TRPV5 and TRPV6 via a kinase-dependent pathway

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12A family of cation-chloride cotransporters and the renal outer medullary K+ channel ROMK and Cl(-) channel SLC26A9. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca2+ channel that serves as a gatekeeper for active Ca2+ reabsorption, WNK3 and TRPV5 were coexpressed in Xenopus laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 82.7 +/- 7.1% increase in TRPV5-mediated Ca2+ uptake was observed when WNK3 was coexpressed. A similar increase in TRPV5-mediated Na+ current was observed with the voltage-clamp technique. WNK3 also enhanced Ca2+ influx and Na+ current mediated by TRPV6, which is the closest homolog of TRPV5 that mediates active intestinal Ca2+ absorption. The kinase domain of WNK3 alone was sufficient to increase TRPV5-mediated Ca2+ transport, and the positive regulatory effect was abolished by the kinase-inactive D294A mutation in WNK3, indicating a kinase-dependent mechanism. The complexly glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3. The exocytosis of TRPV5 was increased by WNK3, and the effect of WNK3 on TRPV5 was abolished by the microtubule inhibitor colchicine. The increased plasma membrane expression of TRPV5 was likely due to the enhanced delivery of mature TRPV5 to the plasma membrane from its intracellular pool via the secretory pathway. These results indicate that WNK3 is a positive regulator of the transcellular Ca2+ transport pathway.

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na+ channel, we examined the effect of Nedd4-2 on the apical Ca2+ channel TRPV6, which is involved in transcellular Ca2+ transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca2+ influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

The A563T variation of the renal epithelial calcium channel TRPV5 among African Americans enhances calcium influx

The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) gene, which encodes the Ca(2+) channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits an unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer (45)Ca(2+) influx assay and the voltage-clamp technique using Xenopus laevis oocytes. Among the variations tested, including A8V, R154H, A563T, and L712F, the latter two significantly increased TRPV5-mediated Ca(2+) influx. The A563T variant, which exists in African Americans with relative high frequency, exhibited increased Ca(2+) influx at extracellular Ca(2+) from 0.01 to 2 mM despite a lower expression level at the plasma membrane. This variant also exhibited a reduction in Na(+) current as a result of increased sensitivity to extracellular Mg(2+). By substituting threonine-563 (Thr(563)) with serine or valine residue, the bulky side chain of Thr(563) was shown to facilitate Ca(2+) transport, whereas the hydroxyl group of Thr(563) is likely related to Mg(2+) sensitivity. The A563T variant was capable of increasing TRPV5-mediated Ca(2+) influx, even when it was expressed under conditions mimicking heterozygous or compound state with other variants. In conclusion, the A563T variant of TRPV5 significantly increased Ca(2+) influx by affecting the Ca(2+) permeation pathway. Thus the A563T variation in TRPV5 may contribute to the superior ability of renal Ca(2+) conservation in African Americans.

WNK4 regulates the secretory pathway via which TRPV5 is targeted to the plasma membrane.

TRPV5 and TRPV6 are two closely related epithelial calcium channels that mediate apical calcium entry in the transcellular calcium transport pathway. TRPV5, but not TRPV6, is enhanced by protein kinase WNK4 when expressed in Xenopus laevis oocytes. We report that the majority of human TRPV5 exogenously expressed in the Xenopus oocyte plasma membrane was complexly N-glycosylated whereas that for human TRPV6 was core-glycosylated. Unglycosylated N358Q mutants of TRPV5 and TRPV6 were able to be expressed in the plasma membrane albeit with decreased abilities in mediating calcium uptake. Syntaxin 6, a SNARE protein in the trans-Golgi network, blocked the complex glycosylation of TRPV5 and TRPV6, rendered the channels in core-glycosylated form. Blocking complex glycosylation of TRPV5 either by syntaxin 6 or by N358Q mutation abolished the enhancing effect of WNK4 on TRPV5. Thus the difference in membrane expression of TRPV5 and TRPV6 explains the selective effect of WNK4 on TRPV5, which is likely on the secretory pathway involving complex glycosylation of channel proteins.

Disease-causing mutations in the acidic motif of WNK4 impair the sensitivity of WNK4 kinase to calcium ions

WNK4 is a serine/threonine protein kinase that is involved in pseudohypoaldosteronism type II (PHAII), a Mendelian form disorder featuring hypertension and hyperkalemia. Most of the PHAII-causing mutations are clustered in an acidic motif rich in negatively charged residues. It is unclear, however, whether these mutations affect the kinase activity in any way. In this study, we isolated kinase domain of WNK4 produced by Escherichia coli, and demonstrated its ability to phosphorylate the oxidative stress-responsive kinase-1 (OSR1) and the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) in vitro. Threonine 48 was identified as the WNK4 phosphorylation site at mouse NCC. The phospho-mimicking T48D mutant of mouse NCC increased its protein abundance and Na(+) uptake, and also enhanced the phosphorylation at the N-terminal region of NCC by OSR1. When the acidic motif was included in the WNK4 kinase construct, the kinase activity of WNK4 exhibited sensitivity to Ca(2+) ions with the highest activity at Ca(2+) concentration around 1 μM using kinase-inactive OSR1 as a substrate. All tested PHAII-causing mutations at the acidic motif exhibited impaired Ca(2+) sensitivity. Our results suggest that these PHAII-causing mutations disrupt a Ca(2+)-sensing mechanism around the acidic motif necessary for the regulation of WNK4 kinase activity by Ca(2+) ions.

Expression and regulation of epithelial Na+ channels by nucleotides in pleural mesothelial cells.

Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and reabsorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that epithelial Na(+) channel (ENaC) is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that three ENaC subunits, namely, alpha, beta, gamma, and two delta ENaC subunits, are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that alpha, beta, gamma, and delta ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride concentration producing 50% inhibition of 12 microM in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (K(m): 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li+ > Na+ > K+. The unitary Li(+) conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na+ channel when coinjected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cyclic AMP and cyclic GMP.

Disease-causing R1185C mutation of WNK4 disrupts a regulatory mechanism involving calmodulin binding and SGK1 phosphorylation sites

The R1185C mutation in WNK4 is associated with pseudohypoaldosteronism type II (PHAII). Unlike other PHAII-causing mutations in the acidic motif, the R1185C mutation is located in the COOH-terminal region of WNK4. The goal of the study is to determine what properties of WNK4 are disrupted by the R1185C mutation. We found that the R1185C mutation is situated in the middle of a calmodulin (CaM) binding site and the mutation reduces the binding of WNK4 to Ca(2+)/CaM. The R1185C mutation is also close to serum- and glucocorticoid-induced protein kinase (SGK1) phosphorylation sites S1190 and S1217. In addition, we identified a novel SGK1 phosphorylation site (S1201) in WNK4, and phosphorylation at this site is reduced by Ca(2+)/CaM. In the wild-type WNK4, the level of phosphorylation at S1190 is the lowest and that at S1217 is the highest. In the R1185C mutant, phosphorylation at S1190 is eliminated and that at S1201 becomes the strongest. The R1185C mutation enhances the positive effect of WNK4 on the Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) as tested in Xenopus laevis oocytes. Deletion of the CaM binding site or phospho-mimicking at two or three of the SGK1 sites enhances the WNK4 effects on NKCC2. These results indicate that the R1185C mutation disrupts an inhibitory domain as part of the suppression mechanism of WNK4, leading to an elevated WNK4 activity at baseline. The presence of CaM binding and SGK1 phosphorylation sites in or close to the inhibitory domain suggests that WNK4 activity is subject to the regulation by intracellular Ca(2+) and phosphorylation.

Suppression of intestinal calcium entry channel TRPV6 by OCRL, a lipid phosphatase associated with Lowe syndrome and Dent disease

Oculocerebrorenal syndrome of Lowe (OCRL) gene product is a phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P(2)] 5-phosphatase, and mutations of OCRL cause Lowe syndrome and Dent disease, both of which are frequently associated with hypercalciuria. Transient receptor potential, vanilloid subfamily, subtype 6 (TRPV6) is an intestinal epithelial Ca(2+) channel mediating active Ca(2+) absorption. Hyperabsorption of Ca(2+) was found in patients of Dent disease with increased Ca(2+) excretion. In this study, we tested whether TRPV6 is regulated by OCRL and, if so, to what extent it is altered by Dent-causing OCRL mutations using Xenopus laevis oocyte expression system. Exogenous OCRL decreased TRPV6-mediated Ca(2+) uptake by regulating the function and trafficking of TRPV6 through different domains of OCRL. The PI(4,5)P(2) 5-phosphatase domain suppressed the TRPV6-mediated Ca(2+) transport likely through regulating the PI(4,5)P(2) level needed for TRPV6 function without affecting TRPV6 protein abundance of TRPV6 at the cell surface. The forward trafficking of TRPV6 was decreased by OCRL. The Rab binding domain in OCRL was involved in regulating the trafficking of TRPV6. Knocking down endogenous X. laevis OCRL by antisense approach increased TRPV6-mediated Ca(2+) transport and TRPV6 forward trafficking. All seven Dent-causing OCRL mutations examined exhibited alleviation of the inhibitory effect on TRPV6-mediated Ca(2+) transport together with decreased overall PI(4,5)P(2) 5-phosphatase activity. In conclusion, OCRL suppresses TRPV6 via two separate mechanisms. The disruption of PI(4,5)P(2) 5-phosphatase activity by Dent-causing mutations of OCRL may lead to increased intestinal Ca(2+) absorption and, in turn, hypercalciuria.

Concerted actions of NHERF2 and WNK4 in regulating TRPV5

With-no-lysine (K) kinase 4 (WNK4) is a protein serine/threonine kinase associated with a Mendelian form of hypertension. WNK4 is an integrative regulator of renal transport of Na(+), K(+), and Cl(-) as shown in Xenopus oocyte system. In addition, WNK4 enhances the surface expression of epithelial Ca(2+) channel TRPV5, which plays a key role in the fine tuning of renal Ca(2+) reabsorption. Variations in the magnitude of WNK4-mediated regulation on TRPV5 in Xenopus oocytes suggest additional cellular components with limited expression are required for the regulation. In this study, we identified the Na(+)/H(+) exchanger regulating factor 2 (NHERF2) as a critical component for the positive regulation of TRPV5 by WNK4. NHERF2 augmented the positive effect of WNK4 on TRPV5, whereas its homolog NHERF1 had no effect when tested in the Xenopus oocyte system. The C-terminal PDZ binding motif of TRPV5 was required for the regulation by NHERF2. While NHERF2 interacted with TRPV5, no association between NHERF2 and WNK4 was detected using a GST pull-down assay. WNK4 increased the forward trafficking of TRPV5; however, it also caused an accelerated decline of the functional TRPV5 channels at later stage of co-expression. NHERF2 stabilized TRPV5 at the plasma membrane without interrupting the forward trafficking of TRPV5, thus prevented the decline of functional TRPV5 channel caused by WNK4 at later stage. The complementary and orderly regulations of WNK4 and NHERF2 allow TRPV5 functions at higher level for a longer period to maximize Ca(2+) influx.