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. 2014 Mar 5;289(16):10999–11006. doi: 10.1074/jbc.M114.553180

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate

THE ROLE OF MEMBRANE ASYMMETRY*

Eric N Senning ‡,1, Marcus D Collins ‡,1, Anastasiia Stratiievska ‡,§,1, Carmen A Ufret-Vincenty ‡, Sharona E Gordon ‡,2
PMCID: PMC4036241  PMID: 24599956

Background: Whether phosphoinositide 4,5-bisphosphate (PI(4,5)P2) activates or inhibits TRPV1 is controversial.

Results: PI(4,5)P2 in the intracellular leaflet activates TRPV1, whereas PI(4,5)P2 in the extracellular leaflet inhibits TRPV1.

Conclusion: Inhibition by PI(4,5)P2 in the extracellular leaflet may explain previous findings that TRPV1 reconstituted into PI(4,5)P2-containing liposomes is inhibited.

Significance: PI(4,5)P2 in the physiologically relevant leaflet (the intracellular leaflet) of the membrane activates TRPV1.

Keywords: Ion Channels; Membrane; Membrane Lipids; Phosphoinositides; Phospholipid; PI(4,5)P2; TRPV1

Abstract

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P2 in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P2 was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P2 whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P2 must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P2 may resolve the long standing controversy about whether PI(4,5)P2 is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.

Introduction

The signaling lipid PI(4,5)P23 is universally accepted to play an essential role in regulation of TRPV1 ion channels. However, whether PI(4,5)P2 activates or inhibits TRPV1 has been debated for over a decade and remains unresolved (2–15). As an activator, PI(4,5)P2 has been proposed to associate with TRPV1 under basal conditions, acting as a cofactor for channel activation. As part of a negative feedback loop in response to Ca2+ influx into a cell, Ca2+ activation of phospholipase C would lead to depletion of PI(4,5)P2 and a consequent reduction in TRPV1 activity. In contrast, PI(4,5)P2 has also been proposed to tonically inhibit TRPV1 under basal conditions. In this scenario, depletion of PI(4,5)P2 by phospholipase C would remove the inhibition, giving an increase in channel activity. This mechanism has been proposed to underlie the potentiation of TRPV1 in response to Gαq-coupled G protein-coupled receptors (5, 6). Interestingly, a dual effect of PI(4,5)P2 has also been proposed in which PI(4,5)P2 potentiates activation at high concentrations of the agonist capsaicin but inhibits activation at low concentrations of capsaicin (6).

The disparate roles proposed for PI(4,5)P2 may arise, at least in part, from different experimental preparations used to study it. In whole cells, PI(4,5)P2 levels can be reduced by converting it to either inositol trisphosphate and diacylglycerol (by phospholipase C) or phosphoinositol 4-phosphate (PI(4)P) by a lipid phosphatase. Do the products of PI(4,5)P2 degradation influence the functional effects observed? We have shown that inositol trisphosphate antagonizes the activating effects of PI(4,5)P2 in excised patches (8), but, of course, this important second messenger can have a variety of targets in intact cells. More recently we have shown that PI(4)P directly activates TRPV1 but that this activation requires at least 20-fold higher concentrations of PI(4)P than PI(4,5)P2 (2, 11) so that it is unlikely to be a factor under typical experimental conditions.

In excised patches from either isolated sensory neurons or cultured cells expressing TRPV1, the activating effect of PI(4,5)P2 on TRPV1 is somewhat less controversial. The application of natural or synthetic PI(4,5)P2 to the intracellular side of inside-out excised patches from cells produces TRPV1 activation in all reported studies (6, 8, 11, 12). One popular form of PI(4,5)P2 used extensively in excised patches is a short-chain, water-soluble synthetic version with octanoyl chains at the sn-1 and sn-2 positions (diC8-PI(4,5)P2, supplemental Fig. 1). Recent characterization of the partition coefficient of diC8-PI(4,5)P2 into bilayers designed to resemble the intracellular leaflet of the plasma membrane allowed the apparent affinity of TRPV1 for diC8-PI(4,5)P2 to be calculated, giving an EC50 of a few tenths of one mole percent (2). Resting levels of PI(4,5)P2 have been measured to be about 1 mol % (16). Although we have not measured the apparent affinity of TRPV1 for natural PI(4,5)P2, it is still unclear whether PI(4,5)P2 levels in the membrane are ever reduced sufficiently to cause its dissociation from TRPV1 under physiological conditions.

In a reduced system in which TRPV1 channels were purified and reconstituted into synthetic vesicles, TRPV1 in excised patches from liposomes prepared with 4 mol % PI(4,5)P2 were shown to be inhibited relative to TRPV1 in patches from liposomes prepared without PI(4,5)P2 (3). Could this inhibition have been due to the extremely high concentration of PI(4,5)P2 in the liposome membrane patches? Or could the structure of the synthetic liposome, compared with the structure of a cell plasma membrane, contribute to PI(4,5)P2 inhibition of TRPV1? Alternatively, is a reevaluation of the effect of PI(4,5)P2 on TRPV1 in excised patches warranted?

We hypothesized that the symmetry of the synthetic liposomes used for studies of reconstituted TRPV1 might explain the observed inhibition by PI(4,5)P2. Cell membranes are asymmetric. That is, their intracellular leaflets and extracellular leaflets are composed of different lipids. Generally, the intracellular leaflet is dominated by phosphatidylethanolamine and phosphatidylserine (PS, about 15 mol %) with about 1 mol % of PI(4,5)P2 and 1 mol % of PI(4)P. The intracellular leaflet, thus, has an overall surface potential of about −25 mV (17). In contrast, the extracellular leaflet is dominated by phosphatidylcholine, with no phosphoinositides (18–20), and has an overall positive charge relative to the intracellular leaflet (16). Thus, membrane proteins see a very different environment in the two leaflets (see Fig. 1). Here, we examined whether this asymmetry matters with respect to regulation of TRPV1 by PI(4,5)P2. We found that PI(4,5)P2 localized to the intracellular leaflet activated TRPV1. Importantly, PI(4,5)P2 symmetrically localized to both leaflets inhibited TRPV1, although this required a very high concentration of PI(4,5)P2 in the extracellular leaflet. These findings may explain the anomalous inhibition of reconstituted TRPV1 channels by PI(4,5)P2 in symmetric liposome patches and emphasize the importance of membrane asymmetry when using reduced systems for studies of membrane proteins.

FIGURE 1.

FIGURE 1.

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Schematic of the relationship between membrane asymmetry and regulation of TRPV1. Blue represents a leaflet of the bilayer that does not contain PI(4,5)P2, and red represents a leaflet of the bilayer that contains PI(4,5)P2.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

HEK 293T/17 cells were cultured at 37 °C, 5% CO2, in Dulbecco's modified Eagle's medium (Invitrogen) containing 25 mm glucose, 1 mm sodium pyruvate, and 4 mm l-glutamine. Culture medium was supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were transfected using Lipofectamine 2000 according to the instructions of the manufacturer. 12 h after transfection, cells were placed on 25-mm glass coverslips (coated with polylysine to aid cell attachment) and left in culture until used for experimentation 12–48 h later.

Electrophysiology Recording

Currents were recorded using filamented borosilicate glass pipettes (1.2-mm outer diameter, 1.0-mm inner diameter) heat polished to a resistance of 3–6 MΩ. Symmetrical solutions were used, containing 130 mm NaCl, 0.2 mm EDTA, and 3 mm HEPES (pH 7.2). Outside-out patches were formed by first using suction to achieve electrical continuity with the cytosol and then slowly moving the pipette away from the cell. Perfusion was achieved by positioning the pipette directly in front of a barrel through which solutions flowed, controlled by an RSC-200 rapid solution changer device (Biologic Instruments). The dioctanoyl phosphoinositides, diC8-PI(4,5)P2, diC8-PI(4)P, diC8-PI (all from Echelon Biosciences), diC8-PS, and diC8-PG (both from Avanti Lipids) were applied in this manner. To prepare aliquots of phospholipids from Echelon Biosciences, we weighed the shipping vials before and after removing the lyophilized product with water to accurately determine the amount supplied to us. Methods to measure TRPV1 current potentiation with diC8-PI(4,5)P2 from inside-out patches have been described previously (11). Inhibition of TRPV1 current in outside-out patches by diC8 phospholipid was determined by calculating a ratio (I/I0) of the current during capsaicin perfusion 30 s after ≥100 s of phospholipid exposure (I) to the current during capsaicin perfusion just prior to phospholipid exposure (I0). GloPI(4,5)P2 (Echelon Biosciences, supplemental Fig. 1) was applied manually as follows. First, most of the solution in the chamber was removed via suction, leaving just enough to immerse the tip of the patch pipette. GloPI(4,5)P2 was then added to the chamber with a glass pipette to an estimated final concentration of 1 μm. This procedure was meant to reduce adherence of the long-chain phosphoinositide to the Teflon and polyethylene perfusion tubing. In all experiments, patches were held at a potential of 0 mV and jumped to +60 mV and −60 mV for between 80–100 ms. Currents shown are difference currents in which the current in the absence of capsaicin has been subtracted. Patches with a seal resistance of <1 GΩ were not used.

Patch Clamp Fluorometry

Experiments were performed using a Nikon TiE microscope equipped with a Photometrics QuantEM camera. Excitation with the 488-nm line of an argon laser was attenuated with an acousto-optic modulator. The emission light was filtered through a 495-nm long-pass filter. Exposure time was 100 ms. Data were analyzed using ImageJ (21). Data shown are the difference in mean intensity within a region of interest including the patch and mean intensity within an adjacent background region.

The fluorescence of gloPI(4,5)P2 was converted to mole fraction of membrane lipids as follows. BODIPY fluorescence self-quenches at high densities because of excimer formation (1) so that small aggregates or vesicles found in solution fluoresce little. As the gloPI(4,5)P2 inserts into the bilayer, it is diluted, and its fluorescence increases. However, as the concentration of gloPI(4,5)P2 in the bilayer increases further, the fluorescence per fluorophore decreases as excimer formation becomes more likely. Above a concentration in the membrane of about 3 mol %, self-quenching dominates, and total fluorescence decreases with increasing concentrations. We measured the fluorescence from gloPI(4,5)P2 in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes at concentrations from 0.5–6.5 mol %. The lipids were mixed in 2:1 chloroform-methanol, dried under a vacuum, resuspended in 155 mm KCl, 25 mm HEPES (pH 7.4) at ∼0.25 mm, and then sonicated in a high-power bath sonicator for 10 min to produce small unilamellar liposomes. 100 μl of each sample and 200 μl of the original buffer were mixed in the wells of a 96-well plate. Fluorescence spectra (500–700 nm) were recorded using 488-nm excitation in a Horiba FluorLog 3 with a plate reader attachment. Fluorescence intensity was integrated from 510–530 nm (to cover the peak which shifts slightly with fluorophore concentration, and this was fit to a model for BODIPY quenching (22), F = QmΓ exp(−πRm2Γ), where Γ is the area density of the fluorophore, Qm accounts for quantum efficiency and geometric factors, and Rm characterizes fluorescence quenching versus distance. The model and data are shown in supplemental Fig. 2a. We found that Rm = 2.6 nm, which is indistinguishable from that for other related BODIPY-labeled lipids (1, 22).

To estimate the mole percent of gloPI(4,5)P2 in the patch membranes, we fit a model to the fluorescence recorded from excised outside-out patches. Fluorescence was calculated from the model parameters (the mole percent of gloPI(4,5)P2 at each time) and the F versus Γ model above. Given the number of parameters, it is necessary to apply a “soft” constraint that the concentration of gloPI(4,5)P2 in the membrane increases at each time step. This is implemented in the fitting function as a finite penalty for the mole fraction decreasing from one time point to the next. The final model fit depends on the strength of the penalty, which is chosen qualitatively. Although the quantification shown is, thus, a rough estimate only, for every choice of the penalty strength we obtain very similar results. The results are shown in Fig. 4 and in more detail in supplemental Fig. 2b. Finally, it should be noted that, although this is a reasonable estimate of mole percent gloPI(4,5)P2 in the membrane, the interaction between gloPI(4,5)P2 and TRPV1 may not have achieved steady state. Therefore, the apparent concentration dependence for inhibition may underestimate the apparent affinity of TRPV1 for gloPI(4,5)P2.

FIGURE 4.

FIGURE 4.

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The fluorescence of gloPI(4,5)P2 reports its concentration in the membrane. Shown is 100 − mol % (blue) and current (black) versus the time from the patch shown in the inset. Calculation of mole percent from fluorescence was as described under “Experimental Procedures.” The experiment was repeated in four patches. Inset, image of an outside-out excised patch in bright-field (grayscale) and epifluorescence (green). Scale bar = 5 μm. cap, capsaicin.

RESULTS

PI(4,5)P2 Activates TRPV1 When in the Intracellular Leaflet of the Plasma Membrane

To test the hypothesis that the asymmetry of PI(4,5)P2 localization in the plasma membrane is important for regulation of TRPV1, we compared the effects of diC8-PI(4,5)P2 (6, 8, 11, 12) on inside-out versus outside-out patches from HEK293T/17 cells transiently transfected with TRPV1. The exogenous agonist capsaicin freely equilibrates across the membrane so that it is equally effective when applied to the extracellular face of an outside-out patch as it is when applied to the intracellular face of an inside-out patch (23). No difference between the patch configurations was observed in the average current amplitude in response to 500 nm capsaicin (inside-out, 1410 pA ± 544 pA, n = 5; outside-out, 1070 pA ± 128 pA, n = 46; p > 0.05).

We have shown previously that TRPV1 is associated with PI(4,5)P2 in inside-out excised patches and that sequestering the PI(4,5)P2 using the nonselective scavenger polylysine leads to dissociation of PI(4,5)P2 from the channels and inhibition of capsaicin-activated current (12). PI(4,5)P2 in the intracellular leaflet of plasma membranes is expected to be at a concentration of roughly 1 mol % (16, 24–26). We have shown recently that a solution concentration of about 50 μm diC8-PI(4,5)P2 is required to give 1 mol % in a synthetic bilayer modeled after the intracellular leaflet of the plasma membrane of dorsal root ganglion neurons (2). As shown in Fig. 2, a and d, perfusion of 50 μm diC8-PI(4,5)P2 onto inside-out patches enhanced TRPV1 activation by capsaicin (after pretreatment of patches with polylysine). Activation of TRPV1 by diC8-PI(4,5)P2 applied to the intracellular leaflet of patches reversed upon removal of diC8-PI(4,5)P2 from the bath (8, 11, 12).

FIGURE 2.

FIGURE 2.

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Opposite regulation of TRPV1 by diC8-PI(4,5)P2 applied to the opposite leaflets of the plasma membrane. Currents during a jump to +60 mV from a holding potential of 0 mV with 500 nm capsaicin (cap) or diC8-PI(4,5)P2 in the bath, as indicated by the red or black bars, respectively. a, example of a current trace from an inside-out patch pretreated with polylysine and then treated with diC8-PI(4,5)P2. b, representative current trace of an outside-out patch treated with diC8-PI(4,5)P2. After two sequential perfusions of 500 nm capsaicin, a solution concentration of 200 μm diC8-PI(4,5)P2 was used for ≥100 s, equivalent to the expected concentration of ≤∼9 mol % in the extracellular leaflet of the membrane (2). c, current trace of outside-out patch with 500 nm capsaicin applied in the presence of a high concentration of >200 μm diC8-PI(4,5)P2. The missing portion of the trace during capsaicin perfusion is due to I-V curve acquisition. Dashed lines represents zero current. d, potentiation or inhibition of TRPV1 currents by application of diC8-PI(4,5)P2 to the intracellular (inside-out, initial capsaicin current (I0) determined just prior to perfusion with diC8-PI(4,5)P2 and final current (I) recorded between 60 and 1135 s after diC8-PI(4,5)P2 was added to the bath, as described in Ref. 11) or extracellular (outside-out, only experiments using perfusion protocol shown in b are included where capsaicin current (I0) determined just prior to perfusion with diC8-PI(4,5)P2 and final current (I) recorded at 30 s after the end of diC8-PI(4,5)P2 perfusion) leaflet. ▿, diC8-PI(4,5)P2 applied to inside-out patch; □, diC8-PI(4,5)P2 applied to outside-out patch, median I/I0 = 0.81 (n = 7, p = 0.016); purple square, I/I0 of representative trace in b. Statistical significance (*) was determined by Wilcoxon signed-rank test, p < 0.05.

PI(4,5)P2 Inhibits TRPV1 When in Both Leaflets of the Plasma Membrane

In excised patches, native PI(4,5)P2 is present only in the intracellular leaflet (i.e. the membrane is asymmetric, Fig. 1). To test the effect of symmetric PI(4,5)P2 in the plasma membrane, we examined the effects of diC8-PI(4,5)P2 applied to the bath on TRPV1 channels in outside-out patches (Fig. 2, b–d). These patches would then have native PI(4,5)P2 in the intracellular leaflet and diC8-PI(4,5)P2 in the extracellular leaflet (Fig. 1). A solution concentration of 50 μm diC8-PI(4,5)P2 did not significantly alter the capsaicin-activated current when applied to outside-out patches. However, application of ≥200 μm diC8-PI(4,5)P2 onto outside-out patches produced robust inhibition of the capsaicin-activated current (Fig. 2, b–d). A solution concentration of 200 μm is expected to produce ≤∼9 mol % in the extracellular leaflet of the patch (2). Interestingly, the recent report showing inhibition of TRPV1 by symmetric PI(4,5)P2 used bilayers with 4 mol % PI(4,5)P2 (3).

An important technical consideration was noted when high concentrations of diC8-PI(4,5)P2 were applied to patches in the presence of ≥500 nm capsaicin. Under these conditions, the effect of diC8-PI(4,5)P2 on the capsaicin-activated current was highly variable (compare Fig. 2, b and c) and not always observed. We suspected that the detergent-like qualities of capsaicin could decrease the critical micellar concentration of diC8-PI(4,5)P2, thereby limiting the concentration of diC8-PI(4,5)P2 monomers in solution and, thus, the mole fraction of diC8-PI(4,5)P2 achieved. On the basis of our previous work, we expect the critical micellar concentration of diC8-PI(4,5)P2 to be greater than ∼3 mm (2). Although this is a factor of 10 or more greater than the concentration of diC8-PI(4,5)P2 used here, it seemed possible that the addition of capsaicin could reduce the free concentration of diC8-PI(4,5)P2 in solution below that required to inhibit TRPV1. Our collected data (Fig. 2d), therefore, reflect only experiments in which diC8-PI(4,5)P2 was applied in the absence of capsaicin. Note that applying diC8-PI(4,5)P2 to outside-out patches and measuring subsequent capsaicin-activated channel activity was only possible because the inhibition produced by diC8-PI(4,5)P2 reversed very slowly upon its removal from the bath (supplemental Fig. 3).

Inhibition of TRPV1 by PI(4,5)P2 in the Extracellular Leaflet Involves PI(4,5)P2 Incorporation into the Bilayer

We next examined whether partitioning of diC8-PI(4,5)P2 into the extracellular leaflet of the plasma membrane was required for its inhibition of TRPV1. Perhaps the headgroup of the phosphoinositide interacted with TRPV1 while in solution without need for its incorporation into the bilayer. A form of PI(4,5)P2 that reports its partitioning into the membrane would allow us to determine whether this is the case.

To determine whether inhibition of TRPV1 by PI(4,5)P2 involves partitioning of PI(4,5)P2 into the extracellular leaflet of the plasma membrane, we used patch clamp fluorometry (27). We used a long-chain PI(4,5)P2, called gloPI(4,5)P2, with a terminal BODIPY-FL moiety at the sn-1 position (supplemental Fig. 1). The properties of the BODIPY-FL moiety are particularly advantageous. Its fluorescence is quenched in aqueous solution, and it becomes fluorescent upon partitioning into the membrane (1) (see “Experimental Procedures”). By recording the BODIPY-FL fluorescence simultaneous with the capsaicin-activated current in an outside-out excised patch, we could determine whether the change in fluorescence we observed tracked the change in current. Of course, for the fluorescence of gloPI(4,5)P2 to be useful, its functional effects must mirror those of diC8-PI(4,5)P2. As shown in Fig. 3, gloPI(4,5)P2 activated TRPV1 when applied to inside-out patches and inhibited TRPV1 when applied to outside-out patches. Thus, gloPI(4,5)P2 is an appropriate surrogate for diC8-PI(4,5)P2, and its fluorescence in patches can be used to measure its partitioning into the extracellular leaflet of the plasma membrane.

FIGURE 3.

FIGURE 3.

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Opposite regulation of TRPV1 by gloPI(4,5)P2 applied to the opposite leaflets of the plasma membrane. Currents measured during a pulse to +60 mV from a holding potential of 0 mV with 500 nm capsaicin (cap) in the absence and presence of 1 μm gloPI(4,5)P2 in the bath, as indicated by the red and black bars, respectively. a, inside-out patch pretreated with polylysine. b, outside-out patch. The dashed line represents zero current. c, collected data from inside-out (n = 4, median I/I0 = 2.1) (▿) and outside-out (n = 8, median I/I0 = 0.39) (□) patches with the current in the presence of gloPI(4,5)P2 (I), determined between 152–529 s after the start of gloPIP2 perfusion depending on the patch, normalized to the current in the absence of gloPI(4,5)P2 (I0), determined just prior to addition of gloPIP2.

As shown in Fig. 4, inset, upon addition of gloPI(4,5)P2 to the patch, the patch fluorescence increased even though little background fluorescence was observed in the bath. We calculated the mole percent of gloPI(4,5)P2 in the patch membrane from the fluorescence in the patch (see supplemental Fig. 2 and “Experimental Procedures”) and plotted one minus mole percent (blue trace) on the same time scale as we plotted the current (black trace). We found that the inhibition of TRPV1 by gloPI(4,5)P2 occurred with the same time course as the partitioning of gloPI(4,5)P2 into the extracellular leaflet of the plasma membrane. The simplest interpretation of the coincidence between partitioning of gloPI(4,5)P2 into the membrane and inhibition of TRPV1 by gloPI(4,5)P2 is that TRPV1 is inhibited by gloPI(4,5)P2 incorporated into the extracellular leaflet of the plasma membrane.

The inhibition of the capsaicin-activated current by gloPI(4,5)P2 became significant above ∼4 mol % (Fig. 4 and supplemental Fig. 2b). This could be an underestimate, however, because the binding of gloPI(4,5)P2 to TRPV1 may not have reached steady state. Even with this caveat, we can conclude that the apparent affinity of TRPV1 for diC8-PI(4,5)P2 (up to between roughly 4 and 9 mol % or between 50 and 200 μm in solution, Fig. 2) and gloPI(4,5)P2 (about 4 mol %, Fig. 4) in the extracellular leaflet are on the same order of magnitude.

Other diC8 Lipids Affect TRPV1 Activity When Applied to the Extracellular Surface of Outside-out Patches

Like PI(4,5)P2, significant amounts of phosphatidylinositol (PI), PI(4)P, and PS are present in the intracellular leaflet of the plasma membrane at rest. PI and PI(4)P are each believed to comprise ∼1 mol % of phospholipids and PS ∼10% of phospholipids in the intracellular leaflet of the plasma membrane (19, 26, 28, 29). In addition, like PI(4,5)P2, PI and PI(4)P have been shown to inhibit TRPV1 reconstituted into liposomes when these phospholipids are present in both leaflets of the bilayer (3). Therefore, we examined whether the addition of diC8-PI, diC8-PI(4)P, diC8-PG, or diC8-PS to outside-out patches would alter the activity of TRPV1 (Fig. 5).

FIGURE 5.

FIGURE 5.

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Representative traces of currents from outside out patches elicited by capsaicin before and after perfusion with diC8 phospholipids. a, 200 μm diC8-PI(4)P treatment. cap, capsaicin. b, 200 μm diC8-PI treatment. c, 200 μm diC8-PS treatment. d, 20 μm diC8-PG treatment. The dashed line represents zero current. e, inhibition of TRPV1 currents by application of different diC8 phospholipids to the extracellular (outside-out, only experiments using perfusion protocol shown in Fig. 2b are included, and calculation of I/I0 is as described for outside-out patches in Fig. 2d) leaflet. □, diC8-PI(4,5)P2, median I/I0 = 0.81 (n = 7, p = 0.016). The purple square is I/I0 of the representative trace in Fig. 2b. ○, diC8-PI(4)P, median I/I0 = 0.95 (n = 10, p = 0.004). ♢, diC8-PI, median I/I0 = 0.99 (n = 7, p = 0.470). ▵, diC8-PS, median I/I0 = 0.83 (n = 6, p = 0.031). Statistical significance (*) or non-significance (NS) of inhibition was determined by Wilcoxon signed-rank test, p < 0.05.

As shown in Fig. 5e, application of 200 μm diC8-PI(4)P and diC8-PS produced a small but statistically significant inhibition of the capsaicin-activated current, whereas application of diC8-PI did not. The experiments with application of diC8-PS showed a broader distribution of responses than the other short-chain phospholipids we tested, and five of 11 patches were lost during the perfusion of diC8-PS (supplemental Fig. 4a). Ordinarily, PS is asymmetrically distributed on the intracellular leaflet of the plasma membrane. However, in a population of cells, we expect that there is some natural variability to the amount of PS that resides in their extracellular leaflets, and this could broaden our distribution of diC8-PS results (29, 30). Addition of 200 μm diC8-PG to outside-out patches resulted in loss of the patch seal in three consecutive experiments (supplemental Fig. 4b), and when we reduced the concentration of diC8-PG to 20 μm, there was no apparent inhibition (n = 3, Fig. 5d). Even when the solution concentrations of each of these short-chain phospholipids were the same, we expect that they would be present in the extracellular leaflet at somewhat higher mole fractions than the cognate concentration of diC8-PI(4,5)P2 because of their lower charge density. We have shown previously that diC8-PI(4)P partitions into a synthetic bilayer modeled to represent the overall charge and saturation of the intracellular leaflet about twice as well as diC8-PI(4,5)P2 (2). Without similar measurements of the partition coefficients of diC8-PI(4)P, diC8-PI, and diC8-PS into synthetic bilayers modeled to represent the overall charge and saturation of the extracellular leaflet, we cannot quantitatively compare the effects of these lipids to those of diC8-PI(4,5)P2.

DISCUSSION

The controversy surrounding whether PI(4,5)P2 in the intracellular leaflet activates or inhibits TRPV1 has important consequences for how we think about its physiological role. Viewed as an activator, PI(4,5)P2 depletion has been proposed to underlie Ca2+-dependent desensitization (6, 7, 9). Viewed as an inhibitor, PI(4,5)P2 depletion has been proposed to underlie G protein-coupled, receptor-mediated sensitization (5). Thus, whether PI(4,5)P2 depletion is activating or inhibitory directs our thinking about whether changes in its concentration produce desensitization or sensitization.

We favor the view that PI(4,5)P2 in the intracellular leaflet of the plasma membrane is an activator of, or at least cofactor for, TRPV1 on the basis of a the following evidence. Application of either short-chain, long-chain, or natural PI(4,5)P2 to the intracellular leaflet of excised patches or to one side of a planar bilayer containing reconstituted TRPV1 potentiates activation of TRPV1(6, 12, 31). Application of purified, recombinant pleckstrin homology domain protein from phospholipase Cδ1, a PI(4,5)P2-selective binding protein, to inside-out excised patches inhibits the capsaicin-activated current (11). Depletion of PI(4,5)P2 by either a voltage-sensitive phosphatase or a chemically inducible phosphatase inhibits capsaicin-activated currents (6, 11). The nonspecific anion-sequestering agent polylysine, which is expected to sequester all anionic lipids in the membrane, inhibits capsaicin-activated currents (6, 12). Inclusion of diC8-PI(4,5)P2 in the whole-cell patch pipette reduces desensitization (32). Ca2+ influx through TRPV1 produces simultaneous PI(4,5)P2 depletion and inhibition of the current (desensitization) (9).

PI(4,5)P2 in the intracellular leaflet has also been proposed to directly inhibit TRPV1. An intriguing new set of findings adds to the evidence for PI(4,5)P2 as an inhibitor of TRPV1. Purified TRPV1 channels were reconstituted into synthetic liposomes of defined composition and analyzed with patch clamp electrophysiology (3). The temperature and capsaicin dependence were measured in liposomes that included either no phosphoinositides or 4% PI(4,5)P2. TRPV1 in the PI(4,5)P2-containing liposomes showed a significantly higher temperature threshold for activation, increasing from about 27 °C (no PI(4,5)P2) to about 38 °C (4% PI(4,5)P2).

A consequence of lipid mixing during liposome production is the symmetric distribution of lipids in the bilayer. In a recent reconstitution experiment with TRPV1, Lukacs et al. generated an asymmetric lipid bilayer by introducing diC8-PI(4,5)P2 to the cis compartment of a painted bilayer (3). These reconstitution experiments convincingly demonstrate that PI(4,5)P2 asymmetry in the plasma membrane is a critical necessity and not inhibitory to TRPV1 function (31).

The inhibition of TRPV1 by PI(4,5)P2 in the extracellular leaflet we report here may reconcile the physiological role of PI(4,5)P2 as an activator of TRPV1 with PI(4,5)P2 inhibition of TRPV1 observed in the reconstituted system of Cao et al. Of course, a number of other experimental differences between our work with channels in biological membranes and observations made using purified channels reconstituted into synthetic bilayers may further contribute to the differences in channel properties measured. However, the PI(4,5)P2 inhibition we report here indicates that the presence of PI(4,5)P2 in both leaflets of the bilayer would be expected to contribute to the PI(4,5)P2 inhibition observed in that system.

Is the inhibition of TRPV1 observed when PI(4,5)P2 is located in both leaflets of the bilayer a general indictment of efforts to reconstitute membrane proteins into membranes of defined composition? We view such well controlled experiments as essential for studies of how membrane properties affect membrane protein function. Nonetheless, the nonphysiological inhibition of TRPV1 produced by symmetric PI(4,5)P2 in synthetic bilayers serves as a reminder that membrane asymmetry matters and challenges us to mimic biologically relevant properties of the membrane when studying reconstituted membrane proteins.

Supplementary Material

Supplemental Data
supp_289_16_10999__index.html (1.1KB, html)

Acknowledgments

We thank Drs. William N. Zagotta, Roger Hardie, and Tamara Rosenbaum for comments on an early version of the manuscript.

*

This work was supported, in whole or in part, by NIGMS, National Institutes of Health Grant R01GM100718; by NEI, National Institutes of Health Grants R01EY017564 and P30EY001730; by the National Center for Research Resources, National Institutes of Health Grant S10RR025429; and by NIDDK, National Institutes of Health Grants P30DK017047, T32EY007031, and T32HL007312.

Inline graphic

This article contains supplemental Figs. 1–4.

3
The abbreviations used are:
PI(4,5)P2
phosphoinositide 4,5-bisphosphate
PI(4)P
phosphoinositol 4-phosphate
PS
phosphatidylserine
PI
phosphatidylinositol.

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Supplementary Materials

Supplemental Data
supp_289_16_10999__index.html (1.1KB, html)
supp_M114.553180_jbc.M114.553180-1.pdf (432KB, pdf)

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