Biophysical Journal Volume 105 December 2013 2485–2494

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Short-Chain Phosphoinositide Partitioning into Plasma Membrane Models Marcus D. Collins† and Sharona E. Gordon†* †

University of Washington School of Medicine, Department of Physiology and Biophysics, Seattle, WA

ABSTRACT Phosphoinositides are vital for many cellular signaling processes, and therefore a number of approaches to manipulating phosphoinositide levels in cells or excised patches of cell membranes have been developed. Among the most common is the use of ‘‘short-chain’’ phosphoinositides, usually dioctanoyl phosphoinositol phosphates. We use isothermal titration calorimetry to determine partitioning of the most abundant phosphoinositol phosphates, PI(4)P and PI(4,5)P2 into models of the intracellular and extracellular facing leaflets of neuronal plasma membranes. We show that phosphoinositide mole fractions in the lipid membrane reach physiological levels at equilibrium with reasonable solution concentrations. Finally we explore the consequences of our results for cellular electrophysiology. In particular, we find that TRPV1 is more selective for PI(4,5)P2 than PI(4) P and activated by extremely low membrane mole fractions of PIPs. We conclude by discussing how the logic of our work extends to other experiments with short-chain phosphoinositides. For delayed rectifier Kþ channels, consideration of the membrane mole fraction of PI(4,5)P2 lipids with different acyl chain lengths suggests a different mechanism for PI(4,5)P2 regulation than previously proposed. Inward rectifier Kþ channels apparent lack of selectivity for certain short-chain PIPs may require reinterpretation in view of the PIPs different membrane partitioning.

INTRODUCTION Phosphoinositol phosphates (PIPs) are key signaling molecules in cells (1–4). These lipids directly modulate ion channel activity, and the products of their hydrolysis, such as diacylglycerol and inositol triphosphate, are key second messengers that regulate numerous cellular functions (5–8). For these reasons, PIPs are intensely studied in neurobiology and related fields. The apparent affinity for PIPs’ regulation of ion channels has been studied using the inside-out patch-clamp technique (4,9–12). Water-soluble PIPs with short acyl chains are often used for reversible delivery into the plasma membrane. Typically, dioctanoyl species are used, such as 1,2-dioctanoyl-sn-glycero-3-phospho-(1’-myo-inositol4’,5’-bisphosphate) (herein diC8-PI(4,5)P2) and 1,2-dioctanoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’-phosphate) (diC8-PI(4)P). Until now, it has not been determined what solution concentration, cphys of each diC8-PIP corresponds to the physiological PIP level in the membrane (1). Even the relative efficacies of diC8-PIPs phosphorylated at different positions (e.g., diC8-PI(4,5)P2 and diC8-PI(4)P) have been inaccessible because, at a given solution concentration, cPIP, the membrane mole fraction of one diC8-PIP, may be different from the other. More precisely, we have not known the partition coefficients Kapp (see Methods for exact definitions) which relate cPIP to mole fractions, XPIP, of diC8-PIPs in the membrane, and are surely different for different diC8PIPs. In addition, effects of diC8-PIPs on ion channels could be because of nonspecific effects if very high membrane

Submitted April 29, 2013, and accepted for publication September 23, 2013. *Correspondence: [email protected]

mole fractions are achieved, especially given the detergent-like nature of these compounds (13). Cell plasma membranes are highly asymmetric, with PIPs localized almost exclusively to the intracellular leaflet (14– 16). In addition, the intracellular leaflet is dominated by PE and PS whereas the extracellular leaflet is dominated by PC (15,17,18). We expect Kapp for different diC8-PIPs to be different for the two leaflets because of their very different overall negative charge, and because of the significantly different spontaneous curvature of PC and PE lipids (19). Determining the Kapp values for diC8-PIPs will facilitate our understanding of the concentration dependence for regulation ion channels and connect experiments in excised patches with experiments in intact cells. In addition, understanding the relationship between cPIP and XPIP for diC8PIPs could help guide experimental design when studying purified ion channels reconstituted into synthetic bilayers. Next we use isothermal titration calorimetry to determine XPIP as a function of cPIP for diC8-PI(4)P in ‘‘intracellular leaflet’’ model bilayers and diC8-PI(4,5)P2 in both ‘‘intracellular leaflet’’ and ‘‘extracellular leaflet’’ model bilayers. Compared with diC8-PI(4)P, XPIP is ~1.7 to 2.5 times smaller for diC8-PI(4,5)P2 for equal solution concentrations, over the range of concentrations typically used in electrophysiology experiments. Using our data we show how to ‘‘correct’’ dose-response relations for activation of the TRPV1 ion channel by diC8-PI(4)P and diC8-PI(4,5) P2 and show that the selectivity for PI(4,5)P2 is even greater than previously appreciated. Not surprisingly, diC8-PI(4,5) P2 partitions more easily into extracellular leaflet models than intracellular leaflet models; this difference is primarily because of the lack of negatively charged lipids in the extracellular leaflet model.

Editor: Heiko Heerklotz. Ó 2013 by the Biophysical Society 0006-3495/13/12/2485/10 $2.00

http://dx.doi.org/10.1016/j.bpj.2013.09.035

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MATERIALS AND METHODS Liposome preparation The ‘‘intracellular’’ leaflet mixture was 21:12:33:33 (molar) of 1,2-dioleoyl-sn-glycerophosphocholine (DOPC), 1-palmitoyl-2-oleoyl-snglycerophosphoserine (POPS), 1,2-dioleoyl-sn-glycero-phosphoethanolamine (DOPE), and cholesterol. The corresponding ‘‘extracellular’’ leaflet mixture was 6:1:3 DOPC:DOPE:cholesterol. All were purchased from Avanti Polar Lipids (Alabaster, AL) as powders and prepared as chloroform stocks. The stocks were then mixed to the final desired concentrations (nominally 50 mM), dried under a dry Argon stream in a clean glass tube, and resuspended in cyclohexane at 50 mg/mL. After freezing at 80 C, lipids in cyclohexane were lyophilized under vacuum until the pressure in the chamber was less than 150 mTorr. The lipids were then resuspended at 50 mM in Buffer A (155 mM KCl, 25 mM HEPES pH 7.4) and extruded through a 200 nm pore filter (GE Healthcare Life Sciences, Piscataway, NJ) using a hand-powered extruder (Avanti Polar Lipids). Liposomes were prepared and used for isothermal titration calorimetry (ITC) experiments on the same day. diC8-PI(4,5)P2 and diC8-PI(4)P (Echelon Biosciences, Salt Lake City, UT) solutions were prepared in Buffer A at 0.6 mM, immediately before the ITC experiments. Buffer was degassed under vacuum for 10 min. After ITC experiments, phosphate concentrations of all stock solutions were determined using a colorometric method (see below). The intracellular and extracellular leaflet mixtures were determined to be 57 mM and 38 mM lipid, respectively, and the two phosphoinositide solutions were both 0.6 mM. The mixtures were also subjected to thin layer chromatography (TLC) to confirm lipid composition.

Isothermal titration calorimetry ITC experiments used a GE Healthcare/MicroCal Auto iTC-200 (GE Healthcare Life Sciences, Piscataway, NJ). In each injection, 3.7 mL of nominally 50 mM liposomes were added to the 200.5 mL calorimetric cell containing the PIP solution. Injections were spaced 6 min apart, except for the highest concentration used in critical micelle concentration (CMC) measurements, when injections were spaced 10 min apart. PIP solutions were prepared immediately before the experiment in the same batch of Buffer A as was used for the liposomes. Baseline subtraction and peak integration were performed automatically by the instrument software. To calculate solution concentrations and account for cell overflow after each injection, we used the dilution model provided by the instrument manufacturer. Extraction of thermodynamic parameters is described in the text. The calorimetric cell contents were recovered for each experiment, and liposomal composition was confirmed after the ITC experiment by TLC (see below). Because of the cost of synthetic phosphoinositides, some care was exercised in planning ITC experiments, especially the measurement of CMC, because they require comparatively large amounts of phosphoinositides compared with typical electrophysiology experiments. To mitigate this cost, we recovered the contents of the calorimetric cell whenever possible to reuse the phosphoinositide solutions. We also omitted the measurement of diC8-PI(4,5)P2’s CMC, because it is only needed to establish that working concentrations for PIP binding experiments are not above the CMC, which is expected to be significantly larger for diC8-PI(4,5)P2 than that for diC8-PI(4)P. Finally, we chose our protocol to measure CMC to minimize the amount of diC8-PI(4)P used, even though other protocols are more standard (20).

Phosphoinositide partitioning and thermal model Although the model used is empirical, in that it makes several assumptions that are likely imperfect, it nonetheless has been useful (20–25). In particular, the model lumps nonideal mixing and any charge effects into an overBiophysical Journal 105(11) 2485–2494

Collins and Gordon all apparent mole-ratio partition coefficient Kapp; we will incorporate charge effects below. A constant ‘‘heat of dilution’’ per injection Qdil, accounts for heats ‘‘from all origins other than solute uptake/release’’ (23), some of which are difficult or impossible to measure directly. The partitioning model is expressed in terms of free (f) and bound (b) PIP concentration, and the total lipid (L) concentration as follows:

R ¼ cbPIP =gcL ¼ Kapp cfPIP

(1)

The parameter g accounts for the ability of the phosphoinositides to reach the inner leaflet of the liposomes. Because large, charged-headgroup lipids are unlikely to translocate (‘‘flip-flop’’) across lipid membranes rapidly, we assume that PIPs incorporate only into the liposomes’ outer leaflet, so that g ¼ 0.5. If the PIPs in fact equilibrate with both leaflets on the timescale of these experiments, the available non-PIP lipid will be doubled (g ¼ 1), whereas our estimate of Kapp would halve. The heat developed for each injection is the sum of the heat of transfer of PIP from solution to liposomes, the true heats of dilution for the liposomes and PIP, and instrumental parameters such as imperfect thermal matching and frictional heat from injection. These are summarized as

QðiÞ ¼ QPIP/L ðiÞ þ Qdil


¼ Kapp gc0PIP ðiÞ c0L ðiÞ  c0L ði  1Þ
2  Vcell DH= 1 þ Kapp gc0L ðiÞ þ Qdil

(2)

where i is the injection number, superscript 0 denotes the total concentration in the calorimetric cell, DH the molar heat of transferring the PIP from solution to the liposomes, and Qdil is a constant heat that summarizes all other contributions. The derivation of this expression, and methods of calculating concentrations after each injection, can be found in the references (21–23). Both phosphoinositides and POPS present in our model liposomes and the cytosolic leaflet of plasma membranes are charged, so we need to explicitly account for charge in our binding model. To do this requires a simple substitution (for examples, see references 22,23,26):

Kapp /K 0 expð  qe ZPIP Dj=kB TÞ

(3)

which replaces an apparent mole-ratio partition coefficient with an ‘‘intrinsic’’ partition coefficient, K0, between phosphoinositides at the membrane-water interface, and those incorporated into the membrane. The exponential factor is a Boltzmann factor relating the phosphoinositide concentration at the interface to that in the bulk solution. Because the surface potential, Dj, and valence, ZPIP, of PIPs are both negative in our case, this factor will always reduce the apparent partition coefficient. Because that valence ZPIP depends indirectly on the surface potential of the lipid membrane (26,27), we constructed a model of the effective charge for diC8-PI(4)P, diC8-PI(4,5)P2, and also POPS that incorporates hydrogen and potassium ion binding. The model is quite tedious, because of the multiple equilibria involved, so the full calculation is presented in the Supporting Material. In brief, we used Toner’s model (26) of ion binding to the 40 and 50 monoester phosphates of PI(4,5)P2, and to the backbone diester phosphate, but for phosphate protonation we used more recent parameters derived from microscopic affinity constants determined by Nuclear Magnetic Resonance (NMR) (27). The key parameters are the intrinsic proton affinity constants, and the affinity for potassium binding to any of the phosphates, assumed to take a common value (26) of 1 M1. For PI(4,5)P2, the proton affinities used (27) are Kp1 ¼ 105.6 M1, Kp2 ¼ 108.3 M1. We assumed that the affinity of protons for the singly protonated 40 and 50 phosphates is negligibly small. Similarly, we take the one proton affinity constant for PI(4)P to be Kp1 ¼ 106.2 M1. We solved the equilibrium equations as a function of surface potential Dj by noting that the affinity constants are multiplied by a factor expðqe ZPIP Dj=kB TÞ to account for the surface potential. Given that the surface potential will in our case always

PIP partition in plasma membrane models

Thin layer chromatography TLC (28) experiments used 10  20 cm EMD/Merck Silica Gel 60 F254 plates. Plates were baked for 1 h at 120 C before use. To facilitate uniform spotting onto the plates, lipids were first extracted from the liposomal suspensions into organic solvents using the method of Folch et al. (29). Standard solutions of POPS, DOPE, and DOPC were prepared in chloroform. An additional lysophosphatidylcholine standard was prepared to examine the samples for degradation. Samples and standards were spotted onto the plate, ~0.75 cm apart on a line 1 cm from one short edge of the plate. The plate was then dried using a hand-held hair dryer. One well of a twowell TLC developing chamber was filled with chloroform/methanol/ammonium hydroxide (65:25:4 by volume), into which a large piece of filter paper was inserted to aid in establishing vapor equilibrium in the chamber. The TLC plate was placed in the dry well, and then the chamber was sealed and allowed to equilibrate for 30 min. Then the chamber was tipped to partially fill the second well, and the TLC plate developed for 30 to 45 min. The plate was removed and air dried, then exposed to iodine vapor for several min. The plate was photographed using a digital camera; densitometry and integration were performed in ImageJ (30). Because unsaturated chains are not labeled by iodine (28), we doubled the integrated result for POPS to determine relative concentrations.

Lipid and short-chain phosphoinositide quantitation To calibrate all solution concentrations, we determined total phosphate in each, using Chen et al.’s colorimetric method (31). All glassware was cleaned prior to use with Nochromix (Godax Laboratories, Cabin John, MD) by soaking for at least 1 h, followed by extensive rinsing in deionized water. Temperatures were continuously monitored to help achieve consistent results. Absorbance was measured at 820 nm. Samples were assayed in triplicate.

RESULTS AND DISCUSSION To increase the experimental value of short-chain PIPs, we determined the relationship between their solution concentration and expected mole fraction in the intracellular leaflet. Inspiration for these experiments came from studies of detergent interactions with lipid membranes. We used isothermal titration calorimetry (ITC) (20,21,23,32) to determine CMC and partitioning of short-chain PIPs into

synthetic lipid membranes designed to mimic the overall composition of the intracellular leaflet of neuronal plasma membranes. Determining the CMC for diC8-PI(4)P To determine reasonable working concentrations for our PIP solutions, we first determined the CMC of diC8-PI(4)P. We used an ‘‘infinite dilution’’ protocol (20): diC8-PI(4)P solutions of different concentrations in Buffer A (see Methods) were titrated into Buffer A. Contents of the calorimetric cell were recovered to be reused in additional titrations as a cost-saving measure (see Methods). The infinite dilution approach allowed us to use concentrations of PIPs at most a few times above the CMC. The conventional approach would have required surfactant in the ITC syringe 10 to 20 times above the CMC; this would have required, for a single experiment, 300 mL of up to 60 mM PI(4)P, making that experiment cost prohibitive. At concentrations above the CMC, one observes a substantial heat of demicellization as a concentrated surfactant is titrated into buffer and the micelles dissociate into monomers; whereas for concentrations below the CMC, one observes only a small heat of dilution. Only a few measurements are needed to obtain an estimate sufficient for our purposes. By fitting lines to the data in the two regimes and finding their intersection, we determined that the diC8PI(4)P CMC is ~3-4 mM (Fig. 1). This value is similar to the 2.3 mM CMC of dioctanoyl-phosphatidylserine (33), which has only a single negative charge. This similarity is likely because the PI(4)P 4’-phosphate is neither fully charged (34) nor as close to the hydrophobic-hydrophilic interface as the backbone phosphate charge shared by phosphatidylserine and PIPs. In contrast, the CMC of dioctanoylphosphoinositol is reported to be 60 mM (35). We did not determine the CMC of diC8-PI(4,5)P2 because the large amount required for measurement was cost prohibitive. However, diC8-PI(4,5)P2’s greater negative charge would

Heat/injection vol., µcal/µL

be negative, it tends to increase local cation concentrations and decrease phosphoinositide and phosphatidylserine valences. With the valences as a function of Dj, we followed the normal procedure (22,23,26) to solve for the surface potential on the lipid membrane as a function of charge density by equating the surface charge density calculated from the lipid composition to that calculated from surface potential and ion screening effects. We took the area per lipid for our diC8-PIPs to be 0.6 nm2, and for all other lipids to be 0.65 nm2. This is something of a guess, as no data are available for the diC8-PIPs. Even for lipids such as DOPE, DOPC, and POPS where data are available (19), areas per lipid are not available for all their combinations; however, using other various reasonable values does not appear to affect the results significantly. As a practical matter to speed calculations, the surface potential was calculated on a grid of values of K0, lipid and phosphoinositide concentrations, which was then interpolated during fitting of the model described by Eqs. 1 to 3 to our data. The process is described in more detail in the Supporting Material.

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0

-10 CMC ~ 3-4 mM

-20

-30

0

1

2

3

4

5

6

7

Titrant diC8-PI(4)P concentration, mM FIGURE 1 Heat of dilution of four solutions of diC8-PI(4)P into buffer. The intersection of the two lines indicates the CMC. Uncertainties in the heat per injection are smaller than the plot symbols. Biophysical Journal 105(11) 2485–2494

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be expected to make its CMC larger than that of diC8-PI(4)P (i.e., > 4 mM).Thus, the following experiments are based on the assumption that the CMC for diC8-PI(4,5)P2 R the CMC for diC8-PI(4)P. Determining Kapp for PI(4)P and PI(4,5)P2 in a model of a plasma membrane ‘‘intracellular’’ leaflet To determine PIPs’ partitioning into the intracellular leaflet of neuronal plasma membranes, we used a ‘‘detergent uptake’’ protocol (23). In this protocol, large unilamellar liposomes formed by extrusion are titrated into a solution containing the PIP of interest below its CMC. We chose a lipid mixture to approximate the lipid composition found in the intracellular leaflet of neuronal cells’ plasma membranes, based on murine synaptosomal plasma membranes (17) and dorsal root ganglia neuronal membranes (36) (Fig. 2, top). This 21:12:33:33 DOPC/POPS/DOPE/cholesterol mixture is notable for its large negative charge (due to POPS) and large quantity of phosphoethanolamine headgroups. Both leaflets of the model liposomes contain the ‘‘intracellular’’ lipid mixture (Fig. 2, bottom). Because charged lipids are often insoluble or poorly soluble in the organic solvents used to prepare lipid mixtures, it is neces-

cell - asymmetric

intracellular leaflet

sary to confirm their presence in liposomes, especially if the solvent is evaporated from a liquid state. We lyophilized our lipids and took care to use appropriate solvents; we nonetheless used TLC to confirm that, even for higher concentrations of POPS, essentially all of the PS found its way into the liposomes used in the ITC experiments (Fig. 3; note that the lipid mixture is slightly different from that used for ITC). This confirmed the mole ratios of lipids in our liposomes were within 10% of the expected values. As PIPs bound to liposomes, the resulting heating power required to thermostat the cell was recorded. The instrument software was used to fit and subtract instrumental background, because of reference heater power, stirring, and similar effects (Fig. 4 a). The software was then used to integrate the peaks, and the data transferred to Mathematica (Wolfram Research, Champaign, IL), to be fit to the partitioning model (described in Methods and references 21,23,25). In separate experiments, the heat of dilution of the liposome solutions into buffer, and buffer into PIP solutions, were measured as checks on the model and instrument, but these data were not used further. To ensure a strong signal, our first experiments used relatively high concentrations (0.6 mM in the ITC cell at the start of each experiment) of diC8-PI(4,5)P2 and diC8PI(4)P. Due presumably to the effects of the large, negative surface charge density, fitting a model having only an apparent, charge-independent, mole ratio partition coefficient Kapp yielded extremely small values for that partition coefficient. The fits were very good (data not shown), but this approach was ultimately unsatisfactory. Although it is not immediately obvious from the equations above, Kapp is

extracellular leaflet

liposomes - symmetric

“intracellular”

“extracellular”

FIGURE 2 Cartoon of intracellular and extracellular leaflets of a cell plasma membrane (top) and of ‘‘intracellular’’ and ‘‘extracellular’’ liposome models (bottom). Biophysical Journal 105(11) 2485–2494

FIGURE 3 TLC of ‘‘intracellular’’ and ‘‘extracellular’’ leaflet-like lipid mixtures. The ‘‘Inner Model’’ (blue curve) was similar but not identical to the intracellular mixture used for ITC; it comprised 1:1:2:2 DOPC: POPS:DOPE:Chol, and integration of the density indicated the final mixture was 0.9:1:2 DOPC:POPS:DOPE (cholesterol was not quantified on the TLC plate). The ‘‘Outer Model’’ of the extracellular leaflet indicated a 5.3:1 ratio of DOPC:DOPE, whereas the expected value was 6:1. Thus, lipid concentrations are within ~10% of the target values. Standards (POPS, DOPC, DOPE) are labeled above the plot and shown as filled regions.

PIP partition in plasma membrane models

a

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a

b b

FIGURE 4 Top panel: Differential heating power measured during serial injections of 57 mM ‘‘intracellular’’ leaflet-like liposomes into 0.6 mM diC8-PI(4,5)P2. Bottom panel: Integrated heats of injection, averaged for three repeats, with standard errors, of the experiment shown in Fig. 1 (top curve) as well as experiments using 0.3 mM (middle curve) and 0.1 mM (bottom curve) diC8-PI(4,5)P2. Solid lines show separate fits of the charge-independent model (21) to data for each concentration of diC8-PI(4,5)P2.

best constrained by the curvature of the heat of injection versus lipid concentration, and by ensuring that nearly all surfactant (i.e., diC8-PI(4)P or diC8-PI(4,5)P2) is bound so that the heat of injection reaches a constant, near-zero value. Thus further experiments were required. To improve the reliability of our model for phosphoinositide binding, we repeated our experiments starting with 0.6 mM, 0.3 mM, or 0.1 mM phosphoinositide in the ITC cell. Again, data collected at each phosphoinositide concentration could be fit very well using a charge-independent Kapp (Fig. 4 b), but as expected the values of Kapp depended on the phosphoinositide concentration. The data could be modeled globally without considering charge effects, but the fit was poor (Fig. 5, dashed lines). Furthermore, the fit was only somewhat improved by assuming a constant valence for PI(4)P, PI(4,5)P2, and POPS in the face of a changing surface potential (data not shown). The best global fit was obtained using the variable charge model (described in Methods), in which the valences are functions of surface potential and hydrogen and potassium ion concentrations. Fig. 5 shows the global, variable-charge model fit (solid lines) to the data for diC8-PI(4)P and diC8-PI(4,5)P2 incorporating into liposomes with a intracellular-leaflet-like composition.

FIGURE 5 Integrated heat for serial injections of intracellular model liposomes into (a) solutions of diC8-PI(4)P or (b) diC8-PI(4,5)P2. In both cases, the phosphoinositide solutions concentrations are, top to bottom, 0.6, 0.3, and 0.1 mM. Dashed lines indicate global fits to the data ignoring the effects of charge; solid lines indicate fits using a variable charge model of the phosphoinositides and POPS. Error bars indicate the standard deviation of three or four repeats.

In electrophysiology experiments we control the unbound solution concentration cf, so we need to know the relationship between solution PIP concentrations and the mole fracb ¼ R=ð1 þ RÞ (see Eq. 1) of each PIP in the tion XPIPx membrane as a function of cf. We found that the partition constant of the PIPs was small but dependent on cf (Fig. 6), because of the charge effects. Up to ~5 mM, we found that Kapp for diC8-PI(4)P is ~1.8 times larger than diC8-PI(4,5)P2: Kapp is 0.64 and 0.36 mM1 for diC8PI(4)P and diC8-PI(4,5)P2, respectively. For comparison, the common detergents octylglucoside and dodecylmaltoside have Kapp values of ~0.2 mM1 and 5 mM1, respectively (21). We note the intrinsic partition coefficients K0 determined from our data (see Methods) are ~6 and 8 mM1 for diC8-PI(4)P and diC8-PI(4,5)P2 respectively; differences in valence between the two PIPs (~1e) coupled to the surface potential (about 20 mV) reduce the partitioning of diC8-PI(4,5)P2 relative to diC8-PI(4)P. Over a wider range of solution concentrations including the highest concentration we typically use for electrophysiology, ~200 mM, no simple function describes the mole fracb of phosphoinositides in the lipid membrane. tion XPIPx However, we have found that we can make a good Biophysical Journal 105(11) 2485–2494

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FIGURE 6 Mole fraction of diC8 phosphoinositides in liposomes modeling the plasma membrane cytosolic leaflet, as a function of free lipid in solution. Values are calculated from the model described in the text, fit to the data shown in Fig. 5. To see this figure in color, go online. b approximation, to ~15% in XPIPx , reasonable for our purposes, by fitting with a polynomial in y ¼ log10 cfPIPx , with solution concentrations expressed in mM: b ¼ 0:00902402y4 þ 0:0812026y3 log10 XPIð4ÞP

þ0:188116y2 þ 0:905069y  0:67812 b log10 XPIð4;5ÞP ¼ 0:00642252y4 þ 0:051329y3 2

þ0:0644365y2 þ 0:685428y  1:07

(4)

(5)

At a solution concentration of 200 mM PI(4)P, as much as is typically tolerated in excised-patch electrophysiology b ~0.06. For PI(4,5)P2 at this experiments, we expect XPIð4ÞP b solution concentration, we expect XPIð4;5ÞP ~0.03. We are 2 also interested in the solution concentration cphys needed to achieve the expected physiological levels of PIPs in the cytosolic leaflet of neuronal plasma membranes, about b ~0.01 (1,2,37). We calculate cphys ~20mM for diC8XPIPx PI(4)P and ~40 mM for diC8-PI(4,5)P2. Some recent ion b ~0.04 channel reconstitution experiments (38) used XPIPx (4 mol %). Using diC8-PIPs to reach this level would require 340 mM diC8-PI(4,5)P2, or 130 mM diC8-PI(4)P, in solution. Such concentrations greatly exceed cphys, raising the question of how to interpret effects observed at nonphysiological PIP concentrations so much higher than achieved in intact cells. Determining Kapp for PI(4,5)P2 in an ‘‘extracellular’’ leaflet model To understand the effect of specific lipids on ion channel function, a reduced system in which the bilayer composition is known and controlled is desirable. For example, one recent study addressed the role of PIPs in regulating TRPV1 ion channels used patch-clamp analysis to measure the function of purified protein reconstituted into synthetic Biophysical Journal 105(11) 2485–2494

liposomes (38). TRPV1 function in liposomes without PIPs was compared with TRPV1 function in liposomes that included 4 mol % of various PIPs. Unlike the experiments shown in Fig. 5, in which PIPs were applied to the intracellular leaflet only, the liposomes used in the reconstitution experiments were symmetric, i.e., PIPs were present in both leaflets of the bilayer. Remarkably, the presence of PIPs in both leaflets of the bilayer produced inhibition of TRPV1—the opposite of what is observed with PIPs present in only the intracellular leaflet (10,12,39–41). To better understand how PIPs alter channel activity in reduced systems, it would be useful to introduce PIPs into only one side of the bilayer, or the other, or both. The water-soluble PIPs described here are ideal for this purpose, as they can be delivered to either leaflet of the bilayer via solution flow onto either ‘‘inside-out’’ or ‘‘outside-out’’ patches. The mechanistic power of this type of experiment motivated us to measure Kapp for PI(4,5)P2 into bilayers modeled after the extracellular leaflet of neuronal plasma membranes. As for ‘‘intracellular’’ leaflet model liposomes, we chose a lipid mixture for the ‘‘extracellular’’ leaflet that mimicked the overall charge, unsaturation, and headgroup distribution of neuronal membrane extracellular leaflets (17,36) (Fig. 2, bottom right). In this case, we used a 6:1:3 molar ratio DOPC/DOPE/cholesterol. Using the same approach as for the ‘‘intracellular’’ leaflet model liposomes, we determined the mole fraction of diC8-PI(4,5)P2 in this model system. Fig. 7 a shows that the fits are not quite as good as for the ‘‘intracellular’’ leaflet model, but they are still adequate. Fig. 7 b compares the mole fraction of diC8-PI(4,5)P2 in extracellular model liposomes with that in cytosolic model liposomes across a range of solution concentrations. In the extracellular model, the mole fraction—and therefore the surface potential—are much larger, and we suspect that the difficulty in fitting the model to our data is because of the limitations of the charge and binding models in these extremes. We have, as above, approximated the mole fraction by the following equation, again in y ¼ log10 cfPIð4;5ÞP2 : b log10 XPIð4;5ÞP ðECÞ ¼ 0:0215878y3 þ 0:0650547y2 2

þ0:418953y  0:706916

(6)

Effect on cellular electrophysiology results To demonstrate how quantifying partitioning of diC8-PI(4)P and diC8-PI(4,5)P2 affects our interpretation of physiological experiments using short-chain PIPs, we have applied our model and measurements to the dose-response relations for activation of TRPV1 ion channels by short-chain PI(4)P and PI(4,5)P2 (10), and to recent work studying the mechanism by which short-chain and natural PI(4,5)P2 potentiates gating of delayed-rectifier potassium channels (42,43). The dose-response relations discussed in both cases are

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FIGURE 8 TRPV1 response to diC8-PI(4,5)P2 (dark gray) or PI(4)P (light gray) in the presence of capsaicin; data from (9). The top axis indicates mole fraction, corresponding to the dashed curves and open symbols. The axis is linearly scaled to best overlay the data for diC8-PI(4,5)P2 onto the corresponding plot versus solution concentration, but no linear transformation does this exactly. Curves indicate Hill equation fits to the data.

FIGURE 7 Extracellular leaflet model. (a) Heats of injection versus lipid concentration for (top to bottom) 0.6, 0.3, and 0.1 mM diC8-PI(4,5)P2. Errors bars represent the larger of the standard deviation for each point, or the root-mean-square of the standard deviations for all points. This was done to avoid unreasonably small weights in the fitting procedure. Dashed lines indicate global fits to the data ignoring the effects of charge; solid lines indicate fits using a variable charge model of the phosphoinositides. (b) Mole fractions of diC8-PI(4,5)P2 versus free concentration in solution for (top dashed curve) the extracellular leaflet model and (bottom solid curve) the intracellular leaflet model.

equilibrium measurements. For instance, Klein et al. (10) excised inside-out patches containing TRPV1 channels from F-11 cells derived from DRG neurons (44), endogenous PIPs were sequestered using poly-lysine, and exogenous short-chain PIPs were applied by perfusion so that the solution concentration (and therefore each PIP’s chemical potential) was fixed. Thus we may apply our equilibrium binding results to dose-response data collected from these inside-out excised patches. The original data and fits with a Hill equation (10) are shown in Fig. 8, overlayed with the same data plotted (open symbols) versus mole fraction of the corresponding phosphoinositide and fitted (dashed lines) with a Hill equation. The upper mole fraction axis is linearly scaled so that the data and fit for diC8-PI(4,5)P2 potentiation of TRPV1 aligns as nearly as possible with the corresponding plot for potentiation versus solution concentration. From Fig. 8 we can draw a few conclusions. First, TRPV1 is more selective for PI(4,5)P2 than was originally thought (10). Although alone this finding affects only the quantitative details of TRPV1, scenarios where the factor of two difference would be important to the qualitative interpretation of the data can be easily imagined. For instance, if a channel

was found have the same response to equal solution concentrations of diC8-PI(4,5)P2 and diC8-PI(4)P, we might have interpreted this to mean that the channel did not bind phosphoinositides with any specificity. However, the higher membrane concentration of diC8-PI(4)P compared with diC8-PI(4,5)P2 under such conditions indicates that diC8PI(4,5)P2 is more effective at producing channel activation, and that the channel may indeed be selective for diC8PI(4,5)P2 over diC8-PI(4)P. Perhaps most importantly, our determination of the relationship between PIPs in solution and in the lipid membrane indicates that TRPV1’s affinity for PIPs is much higher than previously thought. Although we find a Kd ~0.0003 (in mole fraction units) for TRPV1 potentiation by PI(4,5)P2, the resting level in the plasma membrane is b ~0.01, about 30 times higher thought to be about XPIð4;5ÞP 2 (see Fig. 8). Substantial loss of activation would only occur if the PI(4,5)P2 mole fraction was reduced substanb b ~103. Even at XPIð4;5ÞP ~104 there tially below XPIð4;5ÞP 2 2 is still some activation, raising the possibility that even when depleted from the membrane, PI(4,5)P2 may not dissociate from the channel during the typical time a cell is stimulated either in patch-clamp experiments or in vivo. The relationship between solution concentration and membrane mole fraction of PIPs can be used to understand the literature for additional types of channels beyond TRPV1. For example, Kir channels display some selectivity for different PIP headgroups (45). 25 mM diC8-PI(4,5)P2, diC8-PI(3,4)P2, or diC8-PI(3,4,5)P3 were applied to patches from cells expressing one of several Kir channels. Each of four classes of Kir channels studied displayed different apparent selectivity for these different PIPs. For instance, in one class of Kir channels, 25 mM diC8-PI(4,5)P2 in Biophysical Journal 105(11) 2485–2494

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tion and membrane were better known. N-Alcohols of varying length have effects on GLIC (47) suggesting that membrane partition may explain much of the differences between them. Similarly, our reading of the effects of nalcohols on KvAP suggests that they depend on their partition between solution and membrane (48). Fatty acids of different length and saturation have varying effects on TRPV1 (49). Future structure-activity relationship studies involving amphiphilic compounds (for instance, recent SAR studies of TRPV1 antagonists (50,51)) should probably explicitly account for the membrane partition of each compound in determining specificity and mechanism. Our results are limited to the lipid mixtures we have used. Clearly different amounts of charge in the lipid membrane will alter how phosphoinositides partition into the membrane, but changes in the underlying lipid mixture may also alter this partition. For instance, we determined K0 for diC8-PI(4,5)P2 partitioning into an intracellular and an extracellular leaflet model, finding K0 to be ~8 and 20 mM1 respectively. This suggests that substantial variability may be observed in lipid mixtures modeling cells of different types. We have not directly addressed the extent to which membrane partitioning explains differences between experiments performed in different types of cells, or among similar compounds. For example, the solution concentration of capsaicin, itself an amphiphilic compound, needed to activate TRPV1 in HEK293T/17 cells is about 10 times that required in F-11 cells (Fig. 9). In light of our data, we wonder if this is simply a difference in the partition of capsaicin into plasma membranes with possibly very different lipid composition, F-11 cells being derived from DRG neurons (44) and HEK293T/17 cells of unclear origin (52). Neuronal tissues have quite different lipid mixtures from many other cell types (53). Similarly, it seems possible that other amphiphilic ligands that appear highly specific or effective may in fact simply incorporate better into a particular lipid membrane than ‘‘less specific’’ ligands. 1

TRPV1 in F11 cells TRPV1 in HEK cells

I/Imax

solution activated about twice as much current as the same concentration of diC8-PI(3,4,5)P3 in solution. For another type of Kir channels examined, all three PIPs produced the same amount of activation. Based on these data, amino acids that contributed to specificity, or the lack thereof, were identified. For Kir2.1 in particular, four amino acids were identified that could change the selectivity profile of Kir2.1. Furthermore, it was concluded that Kir6.2 was activated in a nonspecific manner by oleoyl-CoA and PIPs. In support of this, Kir6.2 was nonselective with respect to solution concentrations of PIPs, PIP-selective Kir channels were not activated by oleoyl-CoA, and mutations to Kir2.1 making it apparently less selective to PIPs also made it sensitive to oleoyl-CoA. In light of our measurements, the above data can be reinterpreted. At equal solution concentrations, there is roughly twice as much diC8-PI(4)P as diC8-PI(4,5)P2 in lipid membranes; and probably a similar ratio holds for diC8-PI(4,5)P2 and diC8-PI(3,4,5)P3. It seems very likely that Kir6.2 is selective, but for diC8-PI(3,4,5)P3 over other PIPs, because we expect the mole fraction of diC8-PI(3,4,5)P3 to be smaller than that for diC8-PI(4,5)P2 at the solution concentration used. Furthermore, it appears that all of the Kir channels studied are selective for diC8-PI(3,4,5)P3 over diC8-PI(3,4)P2. Thus the conclusion that Kir6.2 and the mutants of Kir2.1 that make it more ‘‘Kir6.2-like’’ are nonselective and discriminate only on charge may need reexamination. Unfortunately, in the absence of full doseresponse curves it is not possible to more precisely define Kir channel specificity. It has been proposed that only the headgroup of PI(4,5)P2 is important to the action of the lipid on delayed-rectifier potassium channels composed of KCNE1/KCNQ1 (42). This conclusion was based on the observation that the same solution concentrations of diC4-PI(4,5)P2, diC8-PI(4,5)P2, or natural cell-extracted PI(4,5)P2 potentiated the channel identically. Based on work by others (20,46) and the data we present in this study, it seems improbable that at equal solution concentration these compounds’ mole fractions in the membrane are identical. Indeed, we expect that at the subsaturating solution concentration used (5 mM in reference 42), the mole fractions for the different PI(4,5)P2 moeities would likely be natural PI(4,5)P2>>diC8-PI(4,5) P2>diC4-PI(4,5)P2. Thus, more natural PI(4,5)P2 would appear to be required for potentiation of KCNE1/KCNQ1 channels than diC8-PI(4,5)P2, and more diC4-PI(4,5)P2 appears to be required than diC8-PI(4,5)P2. This interpretation, that short-chain PIPs appear to be more effective at potentiating KCNE1/KCNQ1 than long-chain PIPs, has no precedent of which we are aware. Alternatively, the channels might interact only with monomeric PI(4,5)P2 in solution, but that would be a curious result given that the channel is sensitive to endogenous PI(4,5)P2 (42). Many studies involving amphiphilic compounds might be better interpreted if the partition coefficients between solu-

Collins and Gordon

0.1

10-8

10-7 Capsaicin, M

10-6

10-5

FIGURE 9 Capsaicin activates TRPV1 at very different concentrations in F11 cells (solid circles; EC50 ~100 nM) and HEK cells (open circles; EC50 ~1mM). Solid lines indicate Hill equation fits.

PIP partition in plasma membrane models

CONCLUSION We have demonstrated that experiments using soluble analogs of physiologically relevant PIPs can be connected to physiological conditions using simple physical chemistry, but our approach is not limited to this special case. A wide variety of natural and synthetic compounds that are used to study ion channels and other membrane proteins are soluble in lipid membranes, but proper comparison of their effects requires knowing how each compound partitions into different lipid membranes.

2493 13. Lundbaek, J. A., P. Birn, ., O. S. Andersen. 2004. Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of micelle-forming amphiphiles and cholesterol. J. Gen. Physiol. 123:599–621. 14. Gascard, P., D. Tran, ., F. Giraud. 1991. Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane. Biochimica. et. Biophysica. Acta. (BBA)–Biomembranes. 1069:27–36. 15. Gascard, P., J. C. Sulpice, ., F. Giraud. 1993. Trans-bilayer distribution of phosphatidylinositol 4,5-bisphosphate and its role in the changes of lipid asymmetry in the human erythrocyte membrane. Biochem. Soc. Trans. 21:253–257.

SUPPORTING MATERIAL

16. Ozato-Sakurai, N., A. Fujita, and T. Fujimoto. 2011. The distribution of phosphatidylinositol 4,5-bisphosphate in acinar cells of rat pancreas revealed with the freeze-fracture replica labeling method. PLoS ONE. 6:e23567.

Model calculations, additional figures and tables are available at http:// www.biophysj.org/biophysj/supplemental/S0006-3495(13)01083-7.

17. Fontaine, R. N., R. A. Harris, and F. Schroeder. 1980. Aminophospholipid asymmetry in murine synaptosomal plasma membrane. J. Neurochem. 34:269–277.

The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ITC experiments were performed with the help of John Sumida at the Analytical Biopharmacy Core of the Center for Intracellular Delivery of Biologics of the University of Washington, which is supported by the Washington State Life Sciences Discovery Fund.

18. Devaux, P. F. 1992. Protein involvement in transmembrane lipid asymmetry. Annu. Rev. Biophys. Biomol. Struct. 21:417–439. 19. Marsh, D. 2013. CRC handbook of lipid bilayers. CRC Press, Boca Raton, FL. 20. Heerklotz, H., and R. M. Epand. 2001. The enthalpy of acyl chain packing and the apparent water-accessible apolar surface area of phospholipids. Biophys. J. 80:271–279.

Research reported in this publication was supported by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM100718 and by the National Eye Institute of the National Institutes of Health under award number R01EY017564.

21. Heerklotz, H., and J. Seelig. 2000. Correlation of membrane/water partition coefficients of detergents with the critical micelle concentration. Biophys. J. 78:2435–2440.

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