Biomedical subjects

Joost G Hoenderop

Publications and source records attributed to Joost G Hoenderop.

6 recordsLinked to original sources

PubMed2006

Extracellular pH dynamically controls cell surface delivery of functional TRPV5 channels.

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.

Animals↗

PubMed2006

Molecular determinants of permeation through the cation channel TRPM6.

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.

Amino Acid Sequence↗

PubMed2006

Critical role of the epithelial Ca2+ channel TRPV5 in active Ca2+ reabsorption as revealed by TRPV5/calbindin-D28K knockout mice.

The epithelial Ca(2+) channel TRPV5 facilitates apical Ca(2+) entry during active Ca(2+) reabsorption in the distal convoluted tubule. In this process, cytosolic Ca(2+) remains at low nontoxic concentrations because the Ca(2+) influx is buffered rapidly by calbindin-D(28K). Subsequently, Ca(2+) that is bound to calbindin-D(28K) is shuttled toward the basolateral Ca(2+) extrusion systems. For addressing the in vivo role of TRPV5 and calbindin-D(28K) in the maintenance of the Ca(2+) balance, single- and double-knockout mice of TRPV5 and calbindin-D(28K) (TRPV5(-/-), calbindin-D(28K)(-/-), and TRPV5(-/-)/calbindin-D(28K)(-/-)) were characterized. These mice strains were fed two Ca(2+) diets (0.02 and 2% wt/wt) to investigate the influence of dietary Ca(2+) content on the Ca(2+) balance. Urine analysis indicated that TRPV5(-/-)/calbindin-D(28K)(-/-) mice exhibit on both diets hypercalciuria compared with wild-type mice. Ca(2+) excretion in TRPV5(-/-)/calbindin-D(28K)(-/-) mice was not significantly different from TRPV5(-/-) mice, whereas calbindin-D(28K)(-/-) mice did not show hypercalciuria. The similarity between TRPV5(-/-)/calbindin-D(28K)(-/-) and TRPV5(-/-) mice was supported further by an equivalent increase in renal calbindin-D(9K) expression and in intestinal Ca(2+) hyperabsorption as a result of upregulation of calbindin-D(9K) and TRPV6 expression in the duodenum. Elevated serum parathyroid hormone and 1,25-dihydroxyvitamin D(3) levels accompanied the enhanced expression of the Ca(2+) transporters. Intestinal Ca(2+) absorption and expression of calbindin-D(9K) and TRPV6, as well as serum parameters of the calbindin-D(28K)(-/-) mice, did not differ from those of wild-type mice. These results underline the gatekeeper function of TRPV5 being the rate-limiting step in active Ca(2+) reabsorption, unlike calbindin-D(28K), which possibly is compensated by calbindin-D(9K).

Absorption↗

PubMed2006

RGS2 inhibits the epithelial Ca2+ channel TRPV6.

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.

Animals↗

PubMed2006

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport.

In Ca(2+)-transporting epithelia, calbindin-D(28K) (CaBP(28K)) facilitates Ca(2+) diffusion from the luminal Ca(2+) entry side of the cell to the basolateral side, where Ca(2+) is extruded into the extracellular compartment. Simultaneously, CaBP(28K) provides protection against toxic high Ca(2+) levels by buffering the cytosolic Ca(2+) concentration ([Ca(2+)](i)) during high Ca(2+) influx. CaBP(28K) consistently colocalizes with the epithelial Ca(2+) channel TRPV5, which constitutes the apical entry step in renal Ca(2+)-transporting epithelial cells. Here, we demonstrate using protein-binding analysis, subcellular fractionation and evanescent-field microscopy that CaBP(28K) translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca(2+)](i). (45)Ca(2+) uptake measurements, electrophysiological recordings and transcellular Ca(2+) transport assays of lentivirus-infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP(28K) tightly buffers the flux of Ca(2+) entering the cell via TRPV5, facilitating high Ca(2+) transport rates by preventing channel inactivation. In summary, CaBP(28K) acts in Ca(2+)-transporting epithelia as a dynamic Ca(2+) buffer, regulating [Ca(2+)] in close vicinity to the TRPV5 pore by direct association with the channel.

Animals↗

PubMed2006

The epithelial Mg2+ channel transient receptor potential melastatin 6 is regulated by dietary Mg2+ content and estrogens.

The kidney is the principal organ responsible for the regulation of the body Mg(2+) balance. Identification of the gene defect in hypomagnesemia with secondary hypocalcemia recently elucidated transient receptor potential melastatin 6 (TRPM6) as the gatekeeper in transepithelial Mg(2+) transport, whereas its homolog, TRPM7, is implicated in cellular Mg(2+) homeostasis. The aim of this study was to determine the tissue distribution in mouse and regulation of TRPM6 and TRPM7 by dietary Mg(2+) and hormones. This study demonstrates that TRPM6 is expressed predominantly in kidney, lung, cecum, and colon, whereas TRPM7 is distributed ubiquitously. Dietary Mg(2+) restriction in mice resulted in hypomagnesemia and renal Mg(2+) and Ca(2+) conservation, whereas a Mg(2+)-enriched diet led to increased urinary Mg(2+) and Ca(2+) excretion. Conversely, Mg(2+) restriction significantly upregulated renal TRPM6 mRNA levels, whereas a Mg(2+) enriched diet increased TRPM6 mRNA expression in colon. Dietary Mg(2+) did not alter TRPM7 mRNA expression in mouse kidney and colon. In addition, it was demonstrated that 17beta-estradiol but not 1,25-dihydroxyvitamin D(3) or parathyroid hormone regulates TRPM6 renal mRNA levels. Renal TRPM7 mRNA abundance remained unaltered under these conditions. The renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17beta-estradiol treatment normalized TRPM6 mRNA levels. In conclusion, kidney, lung, cecum, and colon likely constitute the main sites of active Mg(2+) (re)absorption in the mouse. In addition, Mg(2+) restriction and 17beta-estradiol upregulated renal TRPM6 mRNA levels, whereas a Mg(2+)-enriched diet stimulated TRPM6 mRNA expression in colon, supporting the gatekeeper function of TRPM6 in transepithelial Mg(2+) transport.

Animals↗