Ca2+-dependent desensitization of TRPV2 channels is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate

Abstract

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca(2+)-dependent desensitization, similar to the other TRPVs, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or on coexpression of mutant calmodulin but was sensitive to changes in membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), suggesting a role of membrane PIP(2) in TRPV2 desensitization. Simultaneous confocal imaging and electrophysiological recording of cells expressing TRPV2 and a fluorescent PIP(2)-binding probe demonstrated that TRPV2 desensitization was concomitant with depletion of PIP(2). We conclude that the decrease in PIP(2) levels on channel activation underlies a major component of Ca(2+)-dependent desensitization of TRPV2 and may play a similar role in other TRP channels.

Figure 1.

Diagram of rat TRPV2 and rat TRPV1 primary sequence. The transmembrane domains (S1–S6) are shown in gray and the pore lining domain is shown in white. The N- and C-terminal regions are cytosolic. TRPV2 and TRPV1 ARDs (Jin et al., 2006; Lishko et al., 2007) are shown in blue, regions suggested to interact with CaM (Numazaki et al., 2003; Rosenbaum et al., 2004) are shown in green, and region suggested to interact with ATP (Kwak et al., 2000; Lishko et al., 2007) is shown in red. Regions in the C-terminal domain of TRPV1 suggested to be important for PIP₂ modulation (proximal residues 686-752 and distal residues 777-820) (Prescott and Julius, 2003; Brauchi et al., 2007) and a potential PIP₂ binding site in the C-terminal domain (proximal residues 647-715) of TRPV2 are shown in orange.

Figure 2.

TRPV2 desensitizes in the presence of external Ca²⁺. A, Representative whole-cell recordings at a holding potential of −60 mV from F-11 cells transiently expressing either TRPV2 (top) or TRPV1 (bottom). TRPV2 and TRPV1 currents were evoked by a prolonged exposure (60 s) to 1 mm 2-APB or 100 μm 2-APB, respectively, either in the presence (left current traces) or absence (right current traces) of Ca²⁺. Each current represents a separate cell. A brief agonist stimulus (20 s) separated by 2 min washes with the standard bath solution containing Ca²⁺ is shown as an inset. B, Summary boxplot showing the amount of remaining steady-state current (60 s) relative to the peak response in the absence or presence of Ca²⁺. Boxes encompass the 25th through 75th percentile of the data, the horizontal bar represents the median, and the whiskers extend to the 10th and 90th percentile of the data. The TRPV2 and TRPV1 mean values for current remaining in the presence of Ca²⁺ are 0.2 ± 0.03 and 0.02 ± 0.006 and in the absence of Ca²⁺ are 0.97 ± 0.01 and 0.97 ± 0.02, respectively. Data represent the mean ± SEM from at least five independent experiments. *p < 0.0001.

Figure 3.

TRPV2-ARD does not interact with CaM. Coomassie-stained gel of the six TRPV ARDs showing the amount of each protein that was loaded on the CaM-agarose beads (input lane), and protein bound to CaM-agarose beads in the presence of 2 mm Ca²⁺ (Ca²⁺ lane) or in the presence of 2 mm EGTA with no added Ca²⁺ (EGTA lane). Molecular weights for TRPV-GST are as follows: 76.4 kDa (TRPV1-ARD), 38.3 kDa (TRPV2-ARD), 38.6 kDa (TRPV3-ARD), 37 kDa (TRPV4-ARD), 35.4 kDa (TRPV5-ARD), and 38 kDa (TRPV6-ARD).

Figure 4.

CaM forms a Ca²⁺-dependent complex with a TRPV2 C-terminal region. A, Coomassie-stained gel of TRPV2-C-MBP:CaM complex on affinity purification of the TRPV2-C-MBP fusion protein from bacteria expressing both CaM and TRPV2-C-MBP. B–D, When coexpressed with CaM (B–D, red trace) or mixed together with exogenous CaM (B, blue trace) in the presence of 10 mm Ca²⁺, the TRPV2-C-GFP fusion construct (B, black trace) elute as a higher molecular weight complex from a size exclusion chromatography column. This complex is disrupted by replacement of Ca²⁺ with 10 mm EDTA (C, black trace). D, TRPV2-MBP fusion construct coexpressed with CaM, purified as shown in A, was injected on a size exclusion chromatography column in the absence (red trace) or presence of 1 μm MLCK peptide (green trace), all in the presence of 10 mm Ca²⁺. E, F, When coexpressed with CaM, a proximal C-terminal-C-GFP fusion construct (TRPV2#645-684-C-GFP) eluted as a higher molecular weight complex from a size exclusion column (E, red trace), but a distal C-terminal fusion construct (TRPV2#704-753-C-GFP) did not (F, red trace).

Figure 5.

Inhibiting CaM does not prevent TRPV2 Ca²⁺-dependent desensitization. A, Representative whole-cell TRPV2 currents in control, cells dialyzed with the MLCK peptide, or cells cotransfected with CaM₁₂₃₄. MLCK peptide, at a concentration of 1 μm, was dialyzed through the patch pipette. For MLCK peptide experiments, recordings were performed 5 min after the formation of whole-cell configuration. B, Summary boxplot showing the amount of remaining steady-state current relative to the peak response. The TRPV2 mean value for current remaining in cells cotransfected with CaM₁₂₃₄ is 0.3 ± 0.05 and 0.21 ± 0.04 in cells dialyzed with 1 μm MLCK peptide. TRPV2 mean values for current remaining in the presence and absence of Ca²⁺ reported on Figure 2 are shown for comparison. Data represent the mean ± SEM from at least three independent experiments. *p < 0.0001.

Figure 6.

Ca²⁺ entry through TRPV2 channels induces PIP₂ depletion. F-11 cells were transfected with either TRPV2 and a GFP fused to the PLCδ1-PH domain (GFP-PLCδ1-PH) that is targeted to the plasma membrane through binding to PIP₂ (A) or with GFP-PLCδ1-PH only (B). Shown are confocal images of GFP-PLCδ1-PH fluorescence from representative cells before and after addition of 1 mm 2-APB in the constant presence of Ca²⁺. PIP₂ depletion was assessed by the translocation of the GFP-PLCδ1-PH from the plasma membrane to the cytosol. Scale bar: 5 μm (applies to all images). The right panel shows summary boxplot of normalized fluorescence intensity of GFP in the cytosol for each condition. Data represent the mean ± SEM from at least three independent experiments. *p < 0.0001.

Figure 7.

Depletion of PIP₂ is coincident with TRPV2 desensitization. A, B, A representative imaging and electrophysiology experiment for Ca²⁺-induced PIP₂ depletion in TRPV2 channels. A, GFP-PLCδ1-PH signal in a tsA cell transfected with TRPV2 and GFP-PLCδ1-PH probe. Shown are confocal images for a time point in the current and fluorescence traces shown in B. Scale bar: 5 μm (applies to all images). B, Traces represent the mean fluorescent data (top) from two different cytosolic regions of interest and the current (bottom) from the cell depicted in A with an arrow. Currents (perforated-patch configuration; holding potential, −60 mV) were elicited by 1 mm 2-APB in a Ca²⁺-free solution and changed to a Ca²⁺-containing solution (1.8 mm Ca²⁺) in the continuous presence of the channel agonist to elicit desensitization. An average perfusion exchange delay of 47 s between Ca²⁺-free and Ca²⁺-containing solutions was observed for all the experiments and is shown in this figure (dotted line) for illustration purposes. C, Summary boxplot showing normalized fluorescence intensity of GFP in the cytosol (left) and amount of current inhibition (right) using the same conditions as in B. Values for fluorescence and current were taken at the same time point. The mean value for fluorescent intensity and current remaining in the absence of Ca²⁺ (open boxes) is 0.9 ± 0.02 and 0.97 ± 0.01 and in the presence of Ca²⁺ is 2 ± 0.1 and 0.2 ± 0.03, respectively. Data represent the mean ± SEM from four independent experiments. *p < 0.0002.

Figure 8.

Natural and diC8-PIP₂ rescue inhibition of TRPV2 by PolyK. A, Representative time courses of TRPV2 activation in inside-out excised patches from F-11 cells. A voltage protocol (inset) was used to drive current through the channel and 100 μm 2-APB was applied to the patch to elicit TRPV2 current. PolyK (30 μg/ml) or diC8-PIP₂ (10 μm) were applied as indicated. B, Summary boxplot of the fractional inhibition by PolyK and the restoration by PIP₂. The mean values for current remaining (I/I_initial) after PolyK treatment is 0.2 ± 0.03 and 0.3 ± 0.04 after washing the PolyK from the bath. Data represent the mean ± SEM from at least three independent experiments. *p < 0.0001, significantly different from PolyK wash.