Identification of a novel KLHL3-interacting motif in the C-terminal region of WNK4.

Mutations in with-no-lysine [K] kinase 4 (WNK4) and kelch-like 3 (KLHL3) are linked to pseudohypoaldosteronism type 2 (PHAII, also known as familial hyperkalemic hypertension or Gordon's syndrome). WNK4 is degraded by a ubiquitin E3 ligase with KLHL3 as the substrate adaptor for WNK4. Several PHAII-causing mutations, e.g. those in the acidic motif (AM) of WNK4 and in the Kelch domain of KLHL3, impair the binding between WNK4 and KLHL3. This results in a reduction in WNK4 degradation and an increase in WNK4 activity, leading to PHAII. Although the AM is important in interacting with KLHL3, it is unclear whether this is the only motif in WNK4 responsible for KLHL3-interacting. In this study, a novel motif of WNK4 that is capable of mediating the degradation of the protein by KLHL3 was identified. This C-terminal motif (termed as CM) is located in amino acids 1051-1075 of WNK4 and is rich in negatively charged residues. Both AM and CM responded to the PHAII mutations in the Kelch domain of KLHL3 in a similar manner, but AM is dominant among the two motifs. The presence of this motif likely allows WNK4 protein to respond to the KLHL3-mediated degradation when the AM is dysfunctional due to a PHAII mutation. This may be one of the reasons why PHAII is less severe when WNK4 is mutated compared to KLHL3 is mutated.

Unveiling the Distinct Mechanisms by which Disease-Causing Mutations in the Kelch Domain of KLHL3 Disrupt the Interaction with the Acidic Motif of WNK4 through Molecular Dynamics Simulation.

Kelch-like 3 (KLHL3) is a substrate adaptor of an E3 ubiquitin ligase complex that regulates the degradation of its substrates, including with-no-lysine [K] kinase 4 (WNK4). Mutations in KLHL3 are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. Many PHAII-causing mutations are located in the Kelch domain of KLHL3 that binds with WNK4; however, detailed mechanisms by which these mutations disrupt the binding are not well-understood. In the present study we use molecular dynamics simulations and Western blot analyses to examine the effects of these mutations on the interaction between the Kelch domain of KLHL3 and the acidic motif (AM) of WNK4. The simulation results correlated well with those from Western blot analyses with the exception of the L387P mutation, which led to deregulation of AM degradation by KLHL3 but not recapitulated by simulations. On the basis of the simulation results, a mutation on the binding surface of the Kelch domain affected the Kelch-AM interaction through two major mechanisms: altering the electrostatic potential of the AM binding site and disrupting the Kelch-AM hydrogen bonds. The mutations buried inside the Kelch domain were predicted by our simulations to have no or modest effects on the Kelch-AM interaction. Buried mutations R384Q and S410L disrupted intramolecular hydrogen bonds within the Kelch domain and affected the Kelch-AM interaction indirectly. No significant effect of buried mutation A340V or A494T on the AM degradation or Kelch-AM interaction was observed, implying these mutations may disrupt mechanisms other than Kelch-AM interaction.

The L530R variation associated with recurrent kidney stones impairs the structure and function of TRPV5.

TRPV5 is a Ca2+-selective channel that plays a key role in the reabsorption of Ca2+ ions in the kidney. Recently, a rare L530R variation (rs757494578) of TRPV5 was found to be associated with recurrent kidney stones in a founder population. However, it was unclear to what extent this variation alters the structure and function of TRPV5. To evaluate the function and expression of the TRPV5 variant, Ca2+ uptake in Xenopus oocytes and western blot analysis were performed. The L530R variation abolished the Ca2+ uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with drastic reduction in complex glycosylation. To assess the structural effects of this L530R variation, TRPV5 was modeled based on the crystal structure of TRPV6 and molecular dynamics simulations were carried out. Simulation results showed that the L530R variation disrupts the hydrophobic interaction between L530 and L502, damaging the secondary structure of transmembrane domain 5. The variation also alters its interaction with membrane lipid molecules. Compared to the electroneutral L530, the positively charged R530 residue shifts the surface electrostatic potential towards positive. R530 is attracted to the negatively charged phosphate group rather than the hydrophobic carbon atoms of membrane lipids. This shifts the pore helix where R530 is located and the D542 residue in the Ca2+-selective filter towards the surface of the membrane. These alterations may lead to misfolding of TRPV5, reduction in translocation of the channel to the plasma membrane and/or impaired Ca2+ transport function of the channel, and ultimately disrupt TRPV5-mediated Ca2+ reabsorption.

Molecular Modeling of the Structural and Dynamical Changes in Calcium Channel TRPV5 Induced by the African-Specific A563T Variation.

Transient receptor potential cation channels, vanilloid subfamily, member 5 (TRPV5) plays a key role in active Ca(2+) reabsorption in the kidney. Variations in TRPV5 occur at high frequency in African populations and may contribute to their higher efficiency of Ca(2+) reabsorption. One of the African specific variations, A563T, exhibits increased Ca(2+) transport ability. However, it is unclear how this variation influences the channel pore. On the basis of the structure of TRPV1, a TRPV5 model was generated to simulate the structural and dynamical changes induced by the A563T variation. On the basis of this model, amino acid residue 563 interacts with V540, which is one residue away from the key residue, D542, involved in Ca(2+) selectivity and Mg(2+) blockade. The A563T variation increases secondary structure stability and reduces dynamical motion of D542. In addition, the A563T variation alters the electrostatic potential of the outer surface of the pore. Differences in contact between selective filter residues and residue 563 and in electrostatic potential between the two TRPV5 variants were also observed in another model derived from an alternative alignment in the selective filters between TRPV5 and TRPV1. These findings indicate that the A563T variation induces structural, dynamical, and electrostatic changes in the TRPV5 pore, providing structural insight into the functional alterations associated with the A563T variation.

TRPV5: a Ca(2+) channel for the fine-tuning of Ca(2+) reabsorption.

TRPV5 is one of the two channels in the TRPV family that exhibit high selectivity to Ca(2+) ions. TRPV5 mediates Ca(2+) influx into cells as the first step to transport Ca(2+) across epithelia. The specialized distribution in the distal tubule of the kidney positions TRPV5 as a key player in Ca(2+) reabsorption. The responsiveness in expression and/or activity of TRPV5 to hormones such as 1,25-dihydroxyvitamin D3, parathyroid hormone, estrogen, and testosterone makes TRPV5 suitable for its role in the fine-tuning of Ca(2+) reabsorption. This role is further optimized by the modulation of TRPV5 trafficking and activity via its binding partners; co-expressed proteins; tubular factors such as calbindin-D28k, calmodulin, klotho, uromodulin, and plasmin; extracellular and intracellular factors such as proton, Mg(2+), Ca(2+), and phosphatidylinositol-4,5-bisphosphate; and fluid flow. These regulations allow TRPV5 to adjust its overall activity in response to the body's demand for Ca(2+) and to prevent kidney stone formation. A point mutation in mouse Trpv5 gene leads to hypercalciuria similar to Trpv5 knockout mice, suggesting a possible role of TRPV5 in hypercalciuric disorders in humans. In addition, the single nucleotide polymorphisms in Trpv5 gene prevalently present in African descents may contribute to the efficient renal Ca(2+) reabsorption among African descendants. TRPV5 represents a potential therapeutic target for disorders with altered Ca(2+) homeostasis.

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.

Molecular insights into the structural and dynamical changes of calcium channel TRPV6 induced by its interaction with phosphatidylinositol 4,5-bisphosphate.

Transient receptor potential vanilloid subfamily member 6 (TRPV6) is an epithelial calcium channel that regulates the initial step of the transcellular calcium transport pathway. TRPV6 is expressed in the kidney, intestine, placenta, and other tissues, and the dysregulation of the channel is implicated in several human cancers. It has been reported that phosphatidylinositol 4,5-bisphosphate (PIP2) activates TRPV6 and its close homologue TRPV5; however, the underlying molecular mechanism is less clear. Recently, a structure of rabbit TRPV5 in complex with dioctanoyl (diC8) PIP2, a soluble form of PIP2, was determined by cryo-electron microscopy. Based on this structure, the structural model of human TRPV6 with PIP2 was set up, and then molecular dynamics simulations were performed for TRPV6 with and without PIP2. Simulation results show that the positively charged residues responsible for TRPV5 binding of diC8 PIP2 are conserved in the interactions between TRPV6 and PIP2. The binding of PIP2 to TRPV6 increases the distance between the diagonally opposed residues D542 in the selectivity filter and that between the diagonally opposed M578 residues in the lower gate of TRPV6. A secondary structural analysis reveals that residues M578 in TRPV6 undergo structural and position changes during the binding of PIP2 with TRPV6. In addition, principal component analysis indicates that the binding of PIP2 increases the dynamical motions of both the selectivity filter and the lower gate of TRPV6. These changes induced by PIP2 favor the channel opening. Thus, this study provides a basis for understanding the mechanism underlying the PIP2-induced TRPV6 channel activation.Communicated by Ramaswamy H. Sarma.

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.

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.

Calcium selective channel TRPV6: Structure, function, and implications in health and disease.

Calcium-selective channel TRPV6 (Transient Receptor Potential channel family, Vanilloid subfamily member 6) belongs to the TRP family of cation channels and plays critical roles in transcellular calcium (Ca2+) transport, reuptake of Ca2+ into cells, and maintaining a local low Ca2+ environment for certain biological processes. Recent crystal and cryo-electron microscopy-based structures of TRPV6 have revealed mechanistic insights on how the protein achieves Ca2+ selectivity, permeation, and inactivation by calmodulin. The TRPV6 protein is expressed in a range of epithelial tissues such as the intestine, kidney, placenta, epididymis, and exocrine glands such as the pancreas, prostate and salivary, sweat, and mammary glands. The TRPV6 gene is a direct transcriptional target of the active form of vitamin D and is efficiently regulated to meet the body's need for Ca2+ demand. In addition, TRPV6 is also regulated by the level of dietary Ca2+ and under physiological conditions such as pregnancy and lactation. Genetic models of loss of function in TRPV6 display hypercalciuria, decreased bone marrow density, deficient weight gain, reduced fertility, and in some cases alopecia. The models also reveal that the channel plays an indispensable role in maintaining maternal-fetal Ca2+ transport and low Ca2+ environment in the epididymal lumen that is critical for male fertility. Most recently, loss of function mutations in TRPV6 gene is linked to transient neonatal hyperparathyroidism and early onset chronic pancreatitis. TRPV6 is overexpressed in a wide range of human malignancies and its upregulation is strongly correlated to tumor aggressiveness, metastasis, and poor survival in selected cancers. This review summarizes the current state of knowledge on the expression, structure, biophysical properties, function, polymorphisms, and regulation of TRPV6. The aberrant expression, polymorphisms, and dysfunction of this protein linked to human diseases are also discussed.

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 Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) 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 Ca(2+) 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.

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.

TRPV5 and TRPV6 in transcellular Ca(2+) transport: regulation, gene duplication, and polymorphisms in African populations.

TRPV5 and TRPV6 are unique members of the TRP super family. They are highly selective for Ca(2+) ions with multiple layers of Ca(2+)-dependent inactivation mechanisms, expressed at the apical membrane of Ca(2+) transporting epithelia, and robustly responsive to 1,25-dihydroxivitamin D(3). These features are well suited for their roles as Ca(2+) entry channels in the first step of transcellular Ca(2+) transport pathways, which are involved in intestinal absorption, renal reabsorption of Ca(2+), placental transfer of Ca(2+) to fetus, and many other processes. While TRPV6 is more broadly expressed in a variety of tissues such as esophagus, stomach, small intestine, colon, kidney, placenta, pancreas, prostate, uterus, salivary gland, and sweat gland, TRPV5 expression is relatively restricted to the distal convoluted tubule and connecting tubule of the kidney. There is only one TRPV6-like gene in fish and birds in comparison to both TRPV5 and TRPV6 genes in mammals, indicating TRPV5 gene was likely generated from duplication of TRPV6 gene during the evolution of mammals to meet the needs of complex renal function. TRPV5 and TRPV6 are subjected to vigorous regulations under physiological, pathological, and therapeutic conditions. The elevated TRPV6 level in malignant tumors such as prostate and breast cancers makes it a potential therapeutic target. TRPV6, and to a lesser extent TRPV5, exhibit unusually high levels of single nucleotide polymorphisms (SNPs) in African populations as compared to other populations, indicating TRPV6 gene was under selective pressure during or after humans migrated out of Africa. The SNPs of TRPV6 and TRPV5 likely contribute to the Ca(2+) conservation mechanisms in African populations.

Auto-inhibitory intramolecular S5/S6 interaction in the TRPV6 channel regulates breast cancer cell migration and invasion.

TRPV6, a Ca-selective channel, is abundantly expressed in the placenta, intestine, kidney and bone marrow. TRPV6 is vital to Ca homeostasis and its defective expression or function is linked to transient neonatal hyperparathyroidism, Lowe syndrome/Dent disease, renal stone, osteoporosis and cancers. The fact that the molecular mechanism underlying the function and regulation of TRPV6 is still not well understood hampers, in particular, the understanding of how TRPV6 contributes to breast cancer development. By electrophysiology and Ca imaging in Xenopus oocytes and cancer cells, molecular biology and numerical simulation, here we reveal an intramolecular S5/S6 helix interaction in TRPV6 that is functionally autoinhibitory and is mediated by the R532:D620 bonding. Predicted pathogenic mutation R532Q within S5 disrupts the S5/S6 interaction leading to gain-of-function of the channel, which promotes breast cancer cell progression through strengthening of the TRPV6/PI3K interaction, activation of a PI3K/Akt/GSK-3ß cascade, and up-regulation of epithelial-mesenchymal transition and anti-apoptosis.

8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties.

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.

Autoinhibition of TRPV6 Channel and Regulation by PIP2.

Transient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N and C termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis, and cancer. The TRPV6 function and regulation remain poorly understood. Here we show that the TRPV6 intramolecular S4-S5 linker to C-terminal TRP helix (L/C) and N-terminal pre-S1 helix to TRP helix (N/C) interactions, mediated by Arg470:Trp593 and Trp321:Ile597 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on the L/C interaction but not reversely. Three cationic residues in S5 or C terminus are involved in binding PIP2 to suppress both interactions thereby activating TRPV6. This study reveals "PIP2 - intramolecular interactions" regulatory mechanism of TRPV6 activation-autoinhibition, which will help elucidating the corresponding mechanisms in other TRP channels.

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.

Modeling the structural and dynamical changes of the epithelial calcium channel TRPV5 caused by the A563T variation based on the structure of TRPV6.

TRPV5, transient receptor potential cation channel vanilloid subfamily member 5, is an epithelial Ca2+ channel that plays a key role in the active Ca2+ reabsorption process in the kidney. A single nucleotide polymorphism (SNP) rs4252499 in the TRPV5 gene results in an A563T variation in the sixth transmembrane (TM) domain of TRPV5. Our previous study indicated that this variation increases the Ca2+ transport function of TRPV5. To understand the molecular mechanism, a model of TRPV5 was established based on the newly deposited structure of TRPV6 that has 83.1% amino acid identity with TRPV5 in the modeled region. Computational simulations were performed to study the structural and dynamical differences between the TRPV5 variants with A563 and T563. Consistent with the TRPV1-based simulation, the results indicate that the A563T variation increases the contacts between residues 563 and V540, which is one residue away from the key residue D542 in the Ca2+-selective filter. The variation enhanced the stability of the secondary structure of the pore region, decreased the fluctuation of residues around residue 563, and reduced correlated and anti-correlated motion between monomers. Furthermore, the variation increases the pore radius at the selective filter. These findings were confirmed using simulations based on the recently determined structure of rabbit TRPV5. The simulation results provide an explanation for the observation of enhanced Ca2+ influx in TRPV5 caused by the A563T variation. The A563T variation is an interesting example of how a residue distant from the Ca2+-selective filter influences the Ca2+ transport function of the TRPV5 channel. Communicated by Ramaswamy H. Sarma.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k.

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

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.