Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone

Abstract

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry gate for active Ca(2+) reabsorption in the kidney. Ca(2+) influx through TRPV5 induces rapid channel inactivation, preventing excessive Ca(2+) influx. This inactivation is mediated by the last ∼30 residues of the carboxy (C) terminus of the channel. Since the Ca(2+)-sensing protein calmodulin has been implicated in Ca(2+)-dependent regulation of several TRP channels, the potential role of calmodulin in TRPV5 function was investigated. High-resolution nuclear magnetic resonance (NMR) spectroscopy revealed a Ca(2+)-dependent interaction between calmodulin and a C-terminal fragment of TRPV5 (residues 696 to 729) in which one calmodulin binds two TRPV5 C termini. The TRPV5 residues involved in calmodulin binding were mutated to study the functional consequence of releasing calmodulin from the C terminus. The point mutants TRPV5-W702A and TRPV5-R706E, lacking calmodulin binding, displayed a strongly diminished Ca(2+)-dependent inactivation compared to wild-type TRPV5, as demonstrated by patch clamp analysis. Finally, parathyroid hormone (PTH) induced protein kinase A (PKA)-dependent phosphorylation of residue T709, which diminished calmodulin binding to TRPV5 and thereby enhanced channel open probability. The TRPV5-W702A mutant exhibited a significantly increased channel open probability and was not further stimulated by PTH. Thus, calmodulin negatively modulates TRPV5 activity, which is reversed by PTH-mediated channel phosphorylation.

Fig. 1.

Calmodulin binding to the TRPV5 C terminus. A native polyacrylamide gel, demonstrating interactions between calmodulin (CaM) and hTRPV5^696–729, is shown. Lanes 1 and 7 contain calmodulin in the presence of Ca²⁺ and EGTA, respectively; lanes 2 and 8 contain hTRPV5^696–729 in the presence of Ca²⁺ and EGTA, respectively. The gel demonstrates that in the presence of calcium, calmodulin is forming complexes with hTRPV5^696–729 (lanes 3 to 6 contain calmodulin and hTRPV5^696–729 at different ratios in the presence of Ca²⁺). In the presence of EGTA, no interactions are detected. It should be noted that the proteins in the absence of SDS separate on the basis of their charge-to-mass ratios. Proteins with higher molecular weight may, in fact, run faster than those with the lower molecular weight.

Fig. 2.

In vitro binding of hTRPV5^696–729 to calmodulin. (A) ¹⁵N-¹H-HSQC spectra of hTRPV5^696–729 in the absence of calmodulin (black) and in the presence of 4-fold excess of calmodulin (red). Each peak corresponds to a unique proton attached to a ¹⁵N nucleus. Amino acid residues are marked for which assignment is unambiguous. Nε, nitrogen atom of the side chain amide group R, W, and Q/N. (B) ¹⁵N-¹H-HSQC spectra of calmodulin at various concentrations of the synthetic peptide hTRPV5^696–729. Calmodulin-peptide molar ratios are represented as follows: black, 1:0; red, 1:1 complex; blue, 1:2 complex.

Fig. 3.

Assay of GST-TRPV5 binding to calmodulin. (A) Sequence alignment of calmodulin binding domains in the last part of the TRPV5 C terminus for rabbit (rb) and human species. Note the difference in the numbering of the amino acid positions. The dots indicate the residues involved in the binding of calmodulin, as identified by NMR spectroscopy. (B) Typical blot of precipitation of GST and GST-TRPV5 (GST-V5) in the presence of 1 mM Ca²⁺ or 5 mM EGTA (only TRPV5-WT). Input, total bacterial cell lysate containing TRPV5; CaM-beads, TRPV5 precipitation by calmodulin beads. (C) Quantification of TRPV5-Ca²⁺ (n = 4), TRPV5-EGTA (n = 3), and GST-Ca²⁺ (n = 4) precipitation. The asterisk denotes a significant difference (P < 0.05) from TRPV5-Ca²⁺. (D and E) Representative blot and quantification of TRPV5-WT, -R690E, -H699D, -R700E, -R706E, and -R707E in the presence of 1 mM Ca²⁺ (all conditions, n = 3). (F and G) Representative blot and quantification of TRPV5-WT, -W702A, -L705A, and -L710A (all conditions, n = 4). *, P < 0.05 versus TRPV5-WT.

Fig. 4.

Fura-2 analysis of TRPV5-WT and various point mutants. (A and B) Representative fura-2 traces in arbitrary units (a.u.) of HEK293 cells transiently transfected with TRPV5-WT and the different point mutants. At t = 1.5 min the extracellular Ca²⁺ solution (1.4 mM) was replaced by an EGTA solution (2 mM). At t = 4 min the EGTA solution was again replaced for Ca²⁺ (1.4 mM). (C) Averaged fura-2 levels of TRPV5-WT (n = 36) and TRPV5 mutants R690E (n = 25), H699D (n = 25), R700E (n = 29), W702A (n = 26), L705A (n = 25), R706E (n = 25), R707E (n = 46), and L710A (n = 40) at t = 0. *, P < 0.05 versus TRPV5-WT. Data from at least three independent experiments are shown.

Fig. 5.

TRPV5-W702A and TRPV5-R706E exhibit diminished Ca²⁺-induced inactivation. (A) Averaged traces of inward Ca²⁺ currents measured with 10 mM extracellular Ca²⁺ during a 3-s step to −100 mV from a holding potential of +70 mV in HEK293 cells expressing either TRPV5-WT (dashed line), TRPV5-W702A (dotted line), or TRPV5-R706E (solid line). (B) Average t1 (time after which channel activity is inhibited by 50%) values of TRPV5-WT (n = 13), TRPV5-W702A (n = 10), and TRPV5-R706E (n = 8) depicted in black bars analyzed via mono-exponential fitting. *, P < 0.05 versus TRPV5-WT. (C) I/V profiles of TRPV5-WT (dashed line), TRPV5-W702A (dotted line), or TRPV5-R706E (solid line). Cells were held at +20 mV, and voltage ramps of 450 ms ranging from −100 to +100 mV were applied to measure I/V relations. (D) Average Na⁺ current density at −80 mV of TRPV5-WT (n = 13), TRPV5-W702A (n = 10), and TRPV5-R706E (n = 8).

Fig. 6.

Role of calmodulin in PTH stimulation of TRPV5. (A and B) Representative blot and quantification of GST-TRPV5 precipitation using calmodulin beads. (C) Fura-2 ratio in arbitrary units (a.u.) of HEK293 cells expressing TRPV5-WT (n = 52), -T709A (n = 51), and -W702A (n = 36) upon PTH stimulation (10 nM) at t = 75 s. (D) Statistical analysis of basal fura-2 ratio at t = 0 (control [CTR]) and after PTH stimulation at t = 3 min. *, P < 0.05 versus CTR. Data from at least three independent experiments are shown. CaM, beads.

Fig. 7.

PTH stimulates single-channel activity of TRPV5-WT but not V5-W702A. (A and B) Cell-attached single-channel recordings were made from HEK293 cells expressing TRPV5-WT or TRPV5-W702A. Channel activity was elicited by step potentials varying from −100 to 80 mV for TRPV5-WT (A) and TRPV5-W702A (B). (C) Amplitude histograms were constructed from regions of the single-channel recordings and were fitted by three Gaussian functions corresponding to closed, one open, or two open levels. The averaged calculated slope conductance was 64 ± 7 pS for TRPV5-WT (n = 4) and 69 ± 5 pS for TRPV5-W702A (n = 3). (D) The average NP_o upon PTH stimulation was assessed by averaging min 2 and 3 while the control (CTR) value reflects min 0 to 1. PTH significantly elevates NP_o in TRPV5-WT-expressing cells, whereas it does not influence NP_o in cells expressing TRPV5-W702A. Under control conditions the NP_o of TRPV5-W702A is significantly higher than that of TRPV5-WT. *, P < 0.05 versus TRPV5 CTR. (E and F) Averaged recording of TRPV5-WT (E, n = 5) and TRPV5-W702A (F, n = 6) over time using cell-attached patch clamp (holding potential, −80 mV). NP_o values were the average of 10-s intervals.

Fig. 8.

Model illustrating TRPV5 stimulation by parathyroid hormone. Panel 1 illustrates TRPV5 regulation under basal conditions. Panel 2 displays TRPV5 regulation at low extracellular Ca²⁺ levels. PTH release in blood stimulates the PTH receptor, triggering a cAMP-PKA pathway. This results in the phosphorylation of the TRPV5 C terminus at residue T709. Phosphorylation of T709 diminishes calmodulin (CaM) binding at the last part of the C terminus, increasing channel-open probability and inducing an enlarged TRPV5-mediated Ca²⁺ influx into the cell. Panel 3 demonstrates the situation in which Ca²⁺ homeostasis is reached, and the increased intracellular Ca²⁺ levels inactivate TRPV5 activity, again via the binding of calmodulin. AC, adenylyl cyclase.