Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport

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.

Figure 1

Coordinated expression and direct association of TRPV5 and CaBP_28K. (A) Colocalization of TRPV5 (green) and CaBP_28K (red) in the DCT and CNT. (B) Correlation in TRPV5 and CaBP_28K 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 CaBP_28K 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 CaBP_28K staining of these different cellular regions in 30 cells of six different tubules was calculated as described in Materials and methods. Significant differences in CaBP_28K 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 CaBP_28K 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-CaBP_28K (GST-CaBP_28K) 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

Subcellular localization of CaBP_28K at low intracellular Ca²⁺ concentrations. (A) Cells were transfected with CaBP_28K and TRPV5 (left panel) or CaBP_28K 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 CaBP_28K using anti-Na,K-ATPase and anti-CaBP_28K 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 CaBP_28K and Na,K-ATPase. Representative blots of three independent experiments are shown.

Figure 3

CaBP_28K translocation at low intracellular Ca²⁺ concentrations. (A) TIRF images of a single cell expressing EYFP-CaBP_28K 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-CaBP_28K in EYFP-CaBP_28K- 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-CaBP_28K 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-CaBP_28K-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

Characterization of a Ca²⁺-insensitive CaBP_28K mutant. (A) Wild-type CaBP_28K and CaBP_28KΔEF were fused to GST (left panel) and ⁴⁵Ca²⁺ binding was determined (right panel). (B) [³⁵S]Methionine-labeled, in vitro-translated CaBP_28KΔEF 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 CaBP_28K was incubated in the presence of increasing amounts of non-radioactive in vitro-translated CaBP_28KΔEF 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

Role of CaBP_28K in TRPV5-mediated ⁴⁵Ca²⁺ uptake. EGFP-TRPV5 and CaBP_28K, CaBP_28KΔEF 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 CaBP_28K-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 CaBP_28K and CaBP_28KΔEF in MDCK cells or MDCK cells expressing EGFP-TRPV5 was verified using anti-CaBP_28K antibodies. (D) Ruthenium red-sensitive ⁴⁵Ca²⁺ uptake of MDCK cells stably expressing both (black bars) TRPV5 and CaBP_28K, CaBP_28KΔEF or empty vector and MDCK cells stably expressing (open bars) CaBP_28K, CaBP_28KΔEF or empty vector. Significant differences in ⁴⁵Ca²⁺ uptake are indicated by an asterisk (P<0.05).

Figure 6

Direct influence of CaBP_28K 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 CaBP_28K (+, n=11) or TRPV5 and CaBP_28KΔEF (Δ, n=12). (B) Averaged IC₅₀ for each of the transfections and recordings shown in panel A.

Figure 7

Effect of CaBP_28KΔEF on transcellular Ca²⁺ transport. (A) Effect of lentivirus-mediated overexpression of GFP and GFP-CaBP_28KΔEF 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-CaBP_28KΔEF (upper panel) was assessed with immunoblotting using rabbit anti-GFP antibody and the expression of endogenous CaBP_28K was checked with monoclonal anti-CaBP_28K antibody that does not recognize GFP-CaBP_28KΔEF (lower panel). To check for equal loading, the expression of the endogenously expressed Na,K-ATPase was measured (lower panel).