RESUMEN
Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca(2+)-selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca(2+)-transporting cells, contributing significantly to the overall Ca(2+) balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, (45)Ca(2+) uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular "kiss and linger" interactions with the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Calcio/metabolismo , Perros , Humanos , Concentración de Iones de Hidrógeno , Transporte de Proteínas , Vesículas Transportadoras/metabolismoRESUMEN
BACKGROUND: Kidney stone formation is a major socioeconomic problem in humans, involving pain, recurrent treatment and renal insufficiency. As most renal precipitates contain calcium as a major component, hypercalciuria is the main risk factor for renal stone formation. Different forms of hypercalciuria can be classified, which primarily arise from defects in the main organs involved in calcium homeostasis. A distinction can be made between renal, absorptive and resorptive hypercalciuria, originating from disturbed calcium handling in kidney, intestine and bone, respectively. A positive family history predisposes individuals to an increased risk of stone formation, which strongly indicates the involvement of genetic susceptibility factors. TRPV5 is the renal epithelial calcium channel that is the gatekeeper protein in active calcium reabsorption in the kidney. TRPV5 gene ablation in mice leads to severe hypercalciuria, implying that TRPV5 is an interesting candidate gene for renal hypercalciuria in humans. This study aims to identify and functionally characterize TRPV5 gene aberrations in patients with renal hypercalciuria. METHODS: The TRPV5 coding region and intron-exon boundaries were screened for gene mutations in 20 subjects displaying renal hypercalciuria after which identified non-synonymous polymorphisms were functionally characterized by patch-clamp analysis. Wild-type and TRPV5 channels including polymorphisms were transiently expressed in human embryonic kidney (HEK) 293 cells and functionally characterized by path-clamp analysis. RESULTS: Genotyping TRPV5 in renal hypercalciuria patients revealed three non-synonymous and five synonymous polymorphisms. Electrophysiological characterization of the TRPV5 mutants did not reveal significant functional changes compared to wild-type TRPV5 channel recordings. CONCLUSIONS: In this specific patient cohort, our data do not support a primary role for TRPV5 in the pathogenesis of renal hypercalciuria. However, TRPV5 cannot be excluded as a candidate gene in hypercalciuria.
Asunto(s)
Hipercalciuria/genética , Polimorfismo de Nucleótido Simple , Canales Catiónicos TRPV/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Estudios de Cohortes , Cartilla de ADN/genética , Heterocigoto , Homocigoto , Humanos , Hipercalciuria/complicaciones , Hipercalciuria/metabolismo , Cálculos Renales/etiología , Cálculos Renales/genética , Ratones , Modelos Moleculares , Técnicas de Placa-Clamp , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismoRESUMEN
TRPM6 and its closest relative TRPM7 are members of the Transient Receptor Potential Melastatin (TRPM) subfamily of cation channels and are known to be Mg2+ permeable. By aligning the sequence of the putative TRPM6 pore with the pore sequences of the other subfamily members, we located in the loop between the fifth and the sixth transmembrane domain, a stretch of amino acids residues, 1028GEIDVC1033, as the potential selectivity filter. Two negatively charged residues, E1024 (conserved in TRPM6, TRPM7, TRPM1 and TRPM3) and D1031 (conserved along the entire TRPM subfamily), were identified as important determinants of cation permeation through TRPM6, because neutralization of both residues into an alanine resulted in non-functional channels. Neutralization of E1029 (conserved in TRPM6, TRPM7, TRPM4 and TRPM5) resulted in channels with increased conductance for Ba2+ and Zn2+, decreased ruthenium red sensitivity and larger pore diameter compared to wild-type TRPM6. Changing the residue I1030 into methionine, resulted in channels with lower conductance for Ni2+, decreased sensitivity to ruthenium red block and reduced pore diameter. Thus, these data demonstrate that amino acid residues E1024, I1030 and D1031 are important for channel function and that subtle amino acid variation in the pore region accounts for TRPM6 permeation properties.
Asunto(s)
Magnesio/metabolismo , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/metabolismo , Secuencia de Aminoácidos , Cationes/metabolismo , Línea Celular , Humanos , Transporte Iónico , Datos de Secuencia Molecular , Mutación , Técnicas de Placa-Clamp , Rojo de Rutenio/farmacología , Alineación de Secuencia , Sodio/metabolismo , Canales Catiónicos TRPM/genética , TransfecciónRESUMEN
The extracellular Ca(2+)-sensing receptor (CaR) is a key-player in plasma Ca(2+) homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca(2+) reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca(2+)-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca(2+) reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca(2+) concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR(R185Q). Interestingly, the activity of TRPV6, TRPV5' closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca(2+) influx via a PMA-insensitive PKC isoform pathway.
Asunto(s)
Células Epiteliales/metabolismo , Activación del Canal Iónico , Receptores Sensibles al Calcio/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Humanos , Isoenzimas/metabolismo , Túbulos Renales Distales/citología , Túbulos Renales Distales/efectos de los fármacos , Túbulos Renales Distales/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transporte de Proteínas/efectos de los fármacos , Conejos , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The transient receptor potential vanilloid channels 5 and 6 (TRPV5/6) are the most Ca(2+)-selective channels within the TRP superfamily of ion channels. These epithelial Ca(2+) channels are regulated at different intra- and extracellular sites by the feedback response of Ca(2+) itself, calciotropic hormones, and by TRPV5/6-associated proteins. In the present study, bioinformatics was used to search for novel TRPV5/6-associated genes. By including pull-down assays and functional analysis, Nipsnap1-a hitherto functionally uncharacterized globular protein-was identified as a novel factor involved in the regulation of TRPV6. Electrophysiological recordings revealed that Nipsnap1 abolishes TRPV6 currents. Subsequent biotinylation assays showed that TRPV6 plasma membrane expression did not change in the presence of Nipsnap1, suggesting that TRPV6 inhibition by Nipsnap1 is independently regulated from reduced cell surface channel expression. In addition, semi-quantitative reverse transcriptase PCR and immunohistochemical labeling of Nipsnap1 indicated that Nipsnap1 is expressed in mouse intestinal tissues-where TRPV6 is predominantly expressed-but that it does not co-localize with TRPV5 in the kidney. In conclusion, this study presents the first physiological function of Nipsnap1 as an associated protein inhibiting TRPV6 activity that possibly exerts its effect directly at the plasma membrane.
Asunto(s)
Canales de Calcio/fisiología , Proteínas/fisiología , Canales Catiónicos TRPV/fisiología , Secuencia de Aminoácidos , Canales de Calcio/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Biología Computacional , Electrofisiología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Unión Proteica , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPV/genética , Distribución TisularRESUMEN
PURPOSE OF REVIEW: Recent studies have greatly increased our knowledge concerning the regulation of renal calcium handling. This review focuses on newly identified calciotropic factors present in the pro-urine and the mechanisms by which they control the transient receptor potential channel vanilloid subtype 5 (TRPV5) which forms the gatekeeper of active renal calcium reabsorption. RECENT FINDINGS: The antiaging hormone klotho regulates TRPV5 activity via a novel mechanism modifying its glycosylation status, thereby entrapping the channel at the cell surface. Functional characterization of tissue kallikrein knockout mice revealed that these animals exhibit a pronounced hypercalciuria, comparable to the calcium leak observed in TRPV5 knockout mice. Recently, it has been demonstrated that tissue kallikrein stimulates active calcium reabsorption via the bradykinin receptor type 2 pathway involving protein kinase C-dependent activation of TRPV5. Finally, the extracellular pH appears to act as a dynamic switch controlling cell surface expression of TRPV5. SUMMARY: Unraveling the molecular mechanisms of TRPV5 channel regulation by the antiaging hormone klotho, tissue kallikrein and extracellular pH demonstrated the existence of novel regulatory mechanisms of active calcium reabsorption acting from the tubular lumen.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Homeostasis/fisiología , Riñón/fisiología , Canales Catiónicos TRPV/fisiología , Animales , Glucuronidasa/genética , Glucuronidasa/fisiología , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Proteínas Klotho , Ratones , Modelos Biológicos , Proteína Quinasa C/fisiología , Calicreínas de Tejido/fisiologíaRESUMEN
In the kidney, the epithelial Ca(2+) channel TRPV5 constitutes the apical entry pathway in the process of active Ca(2+) reabsorption. The regulation of Ca(2+) influx through TRPV5 is of crucial importance, because it determines the final amount of Ca(2+) excreted in the urine. The present study identifies FKBP52 as an auxiliary protein of TRPV5, inhibiting channel activity. FKBP52 shows specific interaction with TRPV5, and both proteins colocalize in the distal part of the nephron. On the functional level, FKBP52 decreases Ca(2+) influx through TRPV5 as demonstrated in radioactive (45)Ca(2+) uptake measurements and electrophysiological studies in TRPV5-overexpressing human embryonic kidney 293 cells. On the other hand, gene silencing of FKBP52 or administration of the FKBP52 blocker FK-506 enhances Ca(2+) influx through TRPV5. The inhibitory action of FKBP52 on TRPV5 activity is blunted by mutation of its peptidyl-propyl cis-trans isomerase domain, showing that the FKBP52 catalytic property is critical for channel activity. In conclusion, these results suggest that FKBP52 plays an important role in the regulation of TRPV5 and thus in the process of Ca(2+) reabsorption.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Canales Catiónicos TRPV/fisiología , Proteínas de Unión a Tacrolimus/fisiología , Técnicas de Cultivo de Célula , Electrofisiología , Silenciador del Gen , Humanos , Riñón/citología , Riñón/embriologíaRESUMEN
The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) entry pathway in the process of active Ca(2+) (re)absorption. By yeast two-hybrid and glutathione S-transferase pulldown analysis we identified RGS2 as a novel TRPV6-associated protein. RGS proteins determine the inactivation kinetics of heterotrimeric G-protein-coupled receptor (GPCR) signaling by regulating the GTPase activity of G(alpha) subunits. Here we demonstrate that TRPV6 interacts with the NH(2)-terminal domain of RGS2 in a Ca(2+)-independent fashion and that overexpression of RGS2 reduces the Na(+) and Ca(2+) current of TRPV6 but not that of TRPV5-transfected human embryonic kidney 293 (HEK293) cells. In contrast, overexpression of the deletion mutant DeltaN-RGS2, lacking the NH(2)-terminal domain of RGS2, in TRPV6-expressing HEK293 cells did not show this inhibition. Furthermore, cell surface biotinylation indicated that the inhibitory effect of RGS2 on TRPV6 activity is not mediated by differences in trafficking or retrieval of TRPV6 from the plasma membrane. This effect probably results from the direct interaction between RGS2 and TRPV6, affecting the gating properties of the channel. Finally, the scaffolding protein spinophilin, shown to recruit RGS2 and regulate GPCR-signaling via G(alpha), did not affect RGS2 binding and electrophysiological properties of TRPV6, indicating a GPCR-independent mechanism of TRPV6 regulation by RGS2.
Asunto(s)
Canales de Calcio/metabolismo , Epitelio/metabolismo , Proteínas RGS/fisiología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismo , Animales , Línea Celular , Humanos , RatonesRESUMEN
The transient receptor potential vanilloid 5 (TRPV5) channel determines urinary Ca(2+) excretion, and is therefore critical for Ca(2+) homeostasis. Interestingly, mice lacking the serine protease tissue kallikrein (TK) exhibit robust hypercalciuria comparable to the Ca(2+) leak in TRPV5 knockout mice. Here, we delineated the molecular mechanism through which TK stimulates Ca(2+) reabsorption. Using TRPV5-expressing primary cultures of renal Ca(2+)-transporting epithelial cells, we showed that TK activates Ca(2+) reabsorption. The stimulatory effect of TK was mimicked by bradykinin (BK) and could be reversed by application of JE049, a BK receptor type 2 antagonist. A cell permeable analog of DAG increased TRPV5 activity within 30 min via protein kinase C activation of the channel since mutation of TRPV5 at the putative PKC phosphorylation sites S299 and S654 prevented the stimulatory effect of TK. Cell surface labeling revealed that TK enhances the amount of wild-type TRPV5 channels, but not of the TRPV5 S299A and S654A mutants, at the plasma membrane by delaying its retrieval. In conclusion, TK stimulates Ca(2+) reabsorption via the BK-activated PLC/DAG/PKC pathway and the subsequent stabilization of the TRPV5 channel at the plasma membrane.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Calicreínas/metabolismo , Túbulos Renales/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Canales Catiónicos TRPV/metabolismo , Sustitución de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/farmacología , Bradiquinina/análogos & derivados , Bradiquinina/metabolismo , Bradiquinina/farmacología , Antagonistas del Receptor de Bradiquinina B2 , Canales de Calcio/deficiencia , Canales de Calcio/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Humanos , Túbulos Renales/citología , Ratones , Ratones Noqueados , Mutación Missense , Oximas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Conejos , Receptor de Bradiquinina B2/agonistas , Receptor de Bradiquinina B2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genética , Vasodilatadores/farmacologíaRESUMEN
To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of GFP-TRPV5 in this cell line showed an intracellular granular distribution. Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on plastic supports was threefold higher than in non-transfected cells. Moreover, apical Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on permeable supports was eightfold higher than basolateral Ca2+ uptake, indicating that GFP-TRPV5 is expressed predominantly in the apical membrane. Patch-clamp analysis showed the presence of typical electrophysiological features of GFP-TRPV5, such as inwardly rectifying currents, inhibition by divalent cations and Ca2+-dependent inactivation. Moreover, the TRPV5 inhibitor ruthenium red completely inhibited Ca2+ uptake in GFP-TRPV5-MDCK cells, whereas Ca2+ uptake in non-transfected cells was not inhibited. The characterized GFP-TRPV5-MDCK cell line was used to assess the regulation of TRPV5. The protein kinase C activator phorbol 12-myristate 13-acetate and the cAMP-elevating compounds forskolin/3-isobutyl-1-methylxanthine, 8-Br-cAMP and PGE2 stimulated TRPV5 activity in GFP-TRPV5-MDCK cells by 121+/-7, 79+/-5, 55+/-4 and 61+/-7%, respectively. These compounds did not affect Ca2+ uptake in non-transfected cells. In conclusion, the GFP-TRPV5-MDCK cell line provides a model to specifically study the regulation of TRPV5 activity.