Phospholipase C-mediated regulation of transient receptor potential vanilloid 6 channels: implications in active intestinal Ca2+ transport

Abstract

Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in intestinal Ca(2+) transport. These channels undergo Ca(2+)-induced inactivation. Here we show that Ca(2+) flowing through these channels activates phospholipase C (PLC) leading to the depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) and formation of inositol 1,4,5-trisphosphate in TRPV6-expressing cells. PIP(2) depletion was inhibited by the two structurally different PLC inhibitors 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and edelfosine. Ca(2+)-induced inactivation of TRPV6 was also prevented by the PLC inhibitors in whole-cell patch-clamp experiments. Ca(2+) signals in TRPV6-expressing cells were transient upon restoration of extracellular Ca(2+) but were rendered more sustained by the PLC inhibitors. Finally, intestinal Ca(2+) transport in the everted duodenal sac assay was enhanced by edelfosine. These observations suggest that Ca(2+)-induced inactivation of TRPV6 limits intestinal Ca(2+) absorption and raise the possibility that Ca(2+) absorption can be enhanced pharmacologically by interfering with PLC activation.

Fig. 1.

U73122 inhibits Ca²⁺-induced inactivation of TRPV6 in patch-clamp experiments. Left, representative whole-cell recordings at a holding potential of -60 mV in HEK293 cells expressing TRPV6. Monovalent currents were initiated by the application of divalent-free solution containing 2 mM EGTA (0 Ca²⁺) for 20 s followed by application of 2 mM Ca²⁺. Recordings were performed in cells pretreated with vehicle (DMSO) (A), 3 μM U73343 (B), or 3 μM U73122 (C) for 2 min followed by a 2-min wash. The inhibitors were not present during the measurements. Right, the mean ± S.E.M. of the currents remaining before the addition of 2 mM Ca²⁺ at time points 1, 2, and 3. The data were normalized to the peak current of the first 0 Ca²⁺ pulse.

Fig. 2.

U73122 promotes Ca²⁺ entry into TRPV6-expressing cells. A to C, time courses of increase in fura-2 fluorescence ratio in cells treated with DMSO, U73343 (3 μM), and U73122 (3 μM), respectively. D, the average changes in fluorescence ratio ± S.E.M. for the peak and 200 s after the addition of extracellular Ca²⁺ in cells treated with DMSO (n = 24), U73343 (n = 55), or U73122 (n = 68).

Fig. 3.

U73122 inhibits Ca²⁺-induced PIP₂ hydrolysis in TRPV6-expressing cells. Fluorescence was measured in HEK293 cells expressing TRPV6 and the CFP- and YFP-tagged PLCδ1 PH domains as described under Materials and Methods. Cells were kept in NDF solution for 20 min before the experiment. During the measurement, NDF was replaced with 2 mM Ca²⁺ to induce PIP₂ depletion. Cells were pretreated with DMSO (A), 3 μM U73343 (B), or 3 μM U73343 (C) for 2 min followed by a 2-min wash, and then 2 mM Ca²⁺ was added. The average change in FRET ratio ± S.E.M. was measured and plotted in D for DMSO (n = 8) U73343 (n = 8), or U73122 (n = 10) -treated cells. E, the effect of U73122 and U73343 on [³H]IP₃ production, induced by 2 mM Ca²⁺. Measurements were performed as described under Materials and Methods.

Fig. 4.

Edelfosine inhibits Ca²⁺-induced inactivation of TRPV6. Whole-cell patch-clamp recordings were performed as described in Fig. 1. Cells were preincubated for 20 min with vehicle (DMSO) or 10 μM edelfosine, which was also present during the measurements. A and B, left, time courses of monovalent currents through TRPV6 in control and edelfosine-treated cells, respectively. Right, mean ± S.E.M. of the currents remaining before the addition of 2 mM Ca²⁺. The data were normalized to the peak current of the first 0 Ca²⁺ pulse (n = 8-9). C, left, time courses of Ca²⁺ entry into control and edelfosine (10 μM) preincubated cells. a and b, ratio values at the peak and 150 s after the addition of 2 mM Ca²⁺. The fluorescence ratio values at b divided by the value at a are described on the right for the control and edelfosine-treated cells.

Fig. 5.

Edelfosine inhibits Ca²⁺-induced PIP₂ hydrolysis. Fluorescence was measured in HEK293 cells expressing TRPV6 and the CFP- and YFP-tagged PLCδ1 PH domains as described under Materials and Methods. Time courses of changes in FRET ratio induced by addition of 2 mM Ca²⁺ in cells expressing TRPV6 pretreated with DMSO (A) or 10 μM edelfosine (B). The average change in FRET ratio ± S.E.M. was measured and plotted in C for DMSO- (n = 8) or edelfosine (n = 13)-treated cells.

Fig. 6.

Edelfosine enhances intestinal Ca²⁺ transport. ⁴⁵Ca uptake of everted duodenal sacs was measured as described under Materials and Methods. Duodenal sacs were pretreated with DMSO or 10 μM edelfosine for 20 min, the vehicle or edelfosine was present throughout the 1-h transport measurement. Edelfosine significantly enhanced ⁴⁵Ca transport, n = 7 for DMSO and n = 9 for edelfosine, p < 0.05.