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. 2006 Jul 12;25(13):2978-88.
doi: 10.1038/sj.emboj.7601186. Epub 2006 Jun 8.

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport

Tim T Lambers  1 , Frank Mahieu, Elena Oancea, Louis Hoofd, Frank de Lange, Arjen R Mensenkamp, Thomas Voets, Bernd Nilius, David E Clapham, Joost G Hoenderop, René J Bindels
Affiliations

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport

Tim T Lambers et al. EMBO J. .

Abstract

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.

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Figures

Figure 1
Figure 1
Coordinated expression and direct association of TRPV5 and CaBP28K. (A) Colocalization of TRPV5 (green) and CaBP28K (red) in the DCT and CNT. (B) Correlation in TRPV5 and CaBP28K expression after treatment (1parathyroidectomized (van Abel et al, 2005); 2tacrolimus (Nijenhuis et al, 2005); 3acidose/alkalose (Nijenhuis et al, 2006); 4thiazide (Nijenhuis et al, 2004, 2005); 5calcimemetics (van Abel et al, 2005); 6ovariectomized (van Abel et al, 2005); 7dexamethasone (Nijenhuis et al, 2004); 8ovariectomized+vitamine D3 (van Abel et al, 2002)). R2=0.9068. (C) Localization of CaBP28K in kidney sections of TRPV5−/− and wild-type mice. The epithelial cells were divided in different regions including apical, apical-middle, middle, basolateral-middle and basolateral as indicated. The immuno-positive CaBP28K staining of these different cellular regions in 30 cells of six different tubules was calculated as described in Materials and methods. Significant differences in CaBP28K intensities within the group are indicated by an asterisk. Open bars represent TRPV5%, closed bars represent wild-type. (D) [35S]Methionine-labeled, in vitro-translated CaBP28K was incubated either in the presence (1 mM CaCl2) or absence (5 mM EDTA) of Ca2+, with GST or GST fused to the N- or C-terminus of TRPV5 and TRPV6 immobilized on glutathione-Sepharose 4B beads. Input control (IP) represents 10% of the total pull-down input. (E) Cells were cotransfected with pEBG-CaBP28K (GST-CaBP28K) and pCINeo-TRPV5-IRES-EGFP or pEBG (GST) and pCINeo-TRPV5-IRES-EGFP (control, C). To decrease the [Ca2+]i, cells were treated with BAPTA-AM. Lysates were loaded on glutathione-Sepharose 4B beads, and after extensive washing co-precipitation was investigated by immunoblotting using the guinea-pig anti-TRPV5 antibody (IP=input). The two TRPV5 immuno-positive bands correspond to the core (lower) and glycosylated forms of the protein.
Figure 2
Figure 2
Subcellular localization of CaBP28K at low intracellular Ca2+ concentrations. (A) Cells were transfected with CaBP28K and TRPV5 (left panel) or CaBP28K and empty vector (right panel) and treated with or without BAPTA-AM. Plasma membrane-enriched fractions were probed for the presence of endogenously expressed Na,K-ATPase and exogenously expressed CaBP28K using anti-Na,K-ATPase and anti-CaBP28K antibodies, respectively. NT=non-transfected. (B) Plasma membrane-enriched fractions of primary CNT/CCD cultures, either treated with or without BAPTA-AM, were isolated and probed for the presence of endogenously expressed CaBP28K and Na,K-ATPase. Representative blots of three independent experiments are shown.
Figure 3
Figure 3
CaBP28K translocation at low intracellular Ca2+ concentrations. (A) TIRF images of a single cell expressing EYFP-CaBP28K and TRPV5-ECFP that was treated with (lower panel) or without (upper panel) BAPTA-AM. Scale bar=5 μm. (B) Average time courses of the TIRF signal of EYFP-CaBP28K in EYFP-CaBP28K- and TRPV5-ECFP-expressing cells that were treated either with (▪) or without (Δ) BAPTA-AM (n=7). Significant differences in total EYFP changes after BAPTA-AM treatment (inset) are indicated by an asterisk (P<0.05). (C) TIRF images of a single cell expressing EYFP-CaBP28K that was treated with (lower panel) or without (upper panel) BAPTA-AM. Scale bar=5 μm. (D) Average time courses of the TIRF signal of EYFP-CaBP28K-expressing cells that were treated either with (▪) or without (Δ) BAPTA-AM (n=5). (E) TIRF images of single cells expressing TRPV5-IRES-EGFP treated with (lower panel) or without (upper panel) BAPTA-AM. Scale bar=5 μm. (F) Average time courses of the TIRF signal in TRPV5-IRES-EGFP-expressing cells that were treated either with (▪) or without (Δ) BAPTA-AM (n=5). In all images, a gradient filter was applied such that saturation of TIRF fluorescence turns red and the intensities were measured between 5 and 10% of the visible ‘footprint' of the cell.
Figure 4
Figure 4
Characterization of a Ca2+-insensitive CaBP28K mutant. (A) Wild-type CaBP28K and CaBP28KΔEF were fused to GST (left panel) and 45Ca2+ binding was determined (right panel). (B) [35S]Methionine-labeled, in vitro-translated CaBP28KΔEF was incubated either in the presence (1 mM CaCl2) or absence (5 mM EDTA) of Ca2+, with GST or GST fused to the N- and C-termini of TRPV5 and TRPV6 immobilized on glutathione-Sepharose 4B beads. Input control (IP) represents 10% of the total pull-down input. (C) [35S]Methionine-labeled in vitro-translated CaBP28K was incubated in the presence of increasing amounts of non-radioactive in vitro-translated CaBP28KΔEF with GST or GST fused to the C-termini of TRPV5 immobilized on glutathione-Sepharose 4B beads. This pull-down experiment was performed in the absence of Ca2+ (5 mM EDTA). Input control (IP) represents 10% of the input.
Figure 5
Figure 5
Role of CaBP28K in TRPV5-mediated 45Ca2+ uptake. EGFP-TRPV5 and CaBP28K, CaBP28KΔEF or empty vector were stably expressed in MDCK cells. The expression level of EGFP-tagged TRPV5 as determined using rabbit anti-GFP antibody (A) or flow cytometry analysis (B; black peaks) reveals that the expression of TRPV5 in empty vector- and CaBP28K-expressing cells is equal. The open peak in the three panels indicates background fluorescence in non-transfected cells. The two TRPV5 immuno-positive bands correspond to the core (lower) and glycosylated forms of EGFP-TRPV5. (C) The stable expression of CaBP28K and CaBP28KΔEF in MDCK cells or MDCK cells expressing EGFP-TRPV5 was verified using anti-CaBP28K antibodies. (D) Ruthenium red-sensitive 45Ca2+ uptake of MDCK cells stably expressing both (black bars) TRPV5 and CaBP28K, CaBP28KΔEF or empty vector and MDCK cells stably expressing (open bars) CaBP28K, CaBP28KΔEF or empty vector. Significant differences in 45Ca2+ uptake are indicated by an asterisk (P<0.05).
Figure 6
Figure 6
Direct influence of CaBP28K on the characteristics of TRPV5. (A) Dose–response curves showing the effect of an increasing [Ca2+]i on the normalized Na+ inward current in divalent-free extracellular solution (DVF) of cells expressing TRPV5 (O, n=9), TRPV5 and CaBP28K (+, n=11) or TRPV5 and CaBP28KΔEF (Δ, n=12). (B) Averaged IC50 for each of the transfections and recordings shown in panel A.
Figure 7
Figure 7
Effect of CaBP28KΔEF on transcellular Ca2+ transport. (A) Effect of lentivirus-mediated overexpression of GFP and GFP-CaBP28KΔEF on transcellular Ca2+ transport in primary rabbit CNT/CCD cells. Averaged transcellular Ca2+ transport for each infection is expressed as mean±s.e.m. A 10 μM portion of ruthenium red was added to the apical side of the cell monolayer during the transport assay to estimate the TRPV5-mediated Ca2+ transport. Significant differences as compared to mock-infected cells are indicated by an asterisk (P<0.05). (B) The expression of GFP and GFP-CaBP28KΔEF (upper panel) was assessed with immunoblotting using rabbit anti-GFP antibody and the expression of endogenous CaBP28K was checked with monoclonal anti-CaBP28K antibody that does not recognize GFP-CaBP28KΔEF (lower panel). To check for equal loading, the expression of the endogenously expressed Na,K-ATPase was measured (lower panel).
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