THE JOURNAL 0 1992 by The American Society for Biochemistry and Molecular Biology,

OF BIOLOGICAL CHEMISTRY Inc.

VOl. 267, No . 5, Issue of February 15, pp. 3473-3481,1992 Printed in U.S.A.

Structural and Functional Characterization of an Inositol Polyphosphate Receptor from Cerebellum*

(Received for publication, April 5, 1991)

Christopher C. ChadwickSO, Anthony P. Timermanll, Akitsugu Saito, Martin Mayrleitner 11, Hansgeorg Schindlerl and Sidney Fleischer From the Department of Molecular Bwbgy, Vanderbilt University, Nashville, Tennessee 37235 and the IIZnstitute for Biophysics, Johunnes Kepler University of Linz, A-4040 Linz, Austria

An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three differ- ent polypeptides with M, of 111,000, 102,000, and 62,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds ino- sitol 1,3,4,5-tetrakisphosphate, inositol hexaphos- phate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IPS, and IPS, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 p ~ , respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are P ~ / P N ~ + -5 and PK+/PcI -19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 p~ IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 p~ IP4. IPS and IPS did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.

Hormone interaction with specific plasma membrane recep- tors trigger the G-protein mediated activation of a phospha- tidylinositol specific phospholipase C which generates both inositol 1,4,5-triphosphate (IP3)’ and diacylglyercol. Diacygly- cero1 activates protein kinase C, whereas IP3 serves as a

* This work was supported in part by National Institutes of Health Grant HL32711 (to S. F.), a grant from the Biomedical Research Support Group (to S. F.) administered by Vanderbilt University, and the American Heart Association (Tennessee Affiliate) (to C. C. C.), and Grants S-45/03 and 07 from the Austrian Research Fonds (to M. M. and H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Investigator of the American Heart Association (Tennessee Af- filiate).

5 Present address: Cardiovascular Pharmacology Department, Sterling Drug Inc., 81 Columbia Turnpike, Rensselaer, NY 12144.

ll Recipient of NRSA Postdoctoral Fellowship of ‘the National Institutes of Health.

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP,, inositol 1,3,4,5-tetrakisphosphate; IPS, phytol or inositol hexaphos- phate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonic acid; DTT, dithiothreitol; BSA, bovine serum albu- min; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography.

second messenger to mobilize calcium from intracellular stores. By this mechanism, the generation of IP3 activates numerous Ca2+-dependent processes (1,2).

Characterization of the molecular machinery involved in the IP3-dependent Ca2+ release process has recently been achieved. An IP3 receptor has been isolated from both cere- bellum (3, 4) and smooth muscle (5). Comparison of the smooth muscle IP3 receptor with the IP3 receptor from cere- bellum indicates that the two receptors are similar both structurally and functionally (6). Functional identity of the IP3 receptor as an intracellular Ca2+ release channel has been indicated by incorporation of the cerebellum IP3 receptor into phospholipid vesicles (7, 8) and, more directly, by reconsiti- tution of the smooth muscle IPS receptor into planar lipid bilayers and observing IP3 activated Ca2+ channels (9, 10).

IP3 generated by the IP3 cascade is converted to other inositol polyphosphates by specific kinases and phosphatases. Some of these inositol polyphosphates have also been sug- gested to be involved in intracellular signaling (1, 2, 11). Inositol 1,3,4,5-tetraphosphate (IP4), a metabolite of IP3, has been postulated to act as an intracellular messenger (11-13). In this study, we describe the purification and characteriza- tion of a receptor from cerebellum which bound IP,. After isolation, we found that it bound also IPS and IP3. Reconsti- tution of this inositol polyphosphate receptor preparation into planar bilayers displays potassium channel activity which is selectively modulated by IP,. A preliminary communication has appeared (14).

EXPERIMENTAL PROCEDURES

Materials IPS was purchased from Calbiochem, IP,, was from Boehringer

Mannheim and IPS was from Sigma. [3H]IP3 (17 Ci/mmol), [’HIIP, (17 Ci/mmol), and [3H]IP, (12 Ci/mmol) were from Du Pont-New England Nuclear. CHAPS and heparin-agarose were obtained from Sigma. High and low molecular weight standards for SDS-PAGE were from Bio-Rad. Gel filtration molecular weight standards and phenyl-Sepharose 4B were from Pharmacia LKB Biotechnology Inc. Bovine brain cerebellum was quick-frozen with liquid nitrogen and stored at -80 “C.

Protein Assay Protein determination was performed by the method of Kaplan

(16) or by scanning densitometry of protein bands on Coomassie Blue-R-250 stained SDS-polyacrylamide gels using an automated gel analysis plus image processing system (Technology Resources, Nash- ville, TN). Bovine serum albumin was used as standard.

Electron Microscopy Negative staining of the purified receptor was performed as de-

scribed previously (5).

3473

3474 Purification of an Inositol Polyphosphute Receptor

Methodology Unless indicated otherwise, all solutions contained protease inhib-

itors obtained from Sigma (pepstatin A at 0.5 pg/ml, leupeptin at 0.5 pg/ml, and aprotinin at 0.21 pglml), and the pH of solutions was adjusted at room temperature. All purification steps and binding assays were performed at 4 "C.

Isolation of Bovine Cerebellum Microsomes' Twelve 5-g portions of frozen bovine cerebellum were each thawed

for 10 min in 250 ml of ice-cold buffer A (50 mM Tris-C1 (pH 7.15), 1 mM EDTA, 1 mM DTT) contained in Beckman JA-10 centrifuge bottles and homogenized for 15 s at setting 6 using a polytron homogenizer (PT 20). The homogenate5 were then centrifuged for 1 h at 14,000 rpm using a Beckman JA-14 rotor. The clear supernatants were discarded and the pellets resuspended in 250 ml buffer A containing 150 mM NaC1. The resuspensions were homogenized for 10 s at setting 6 using the polytron and centrifuged as described above. The pellets were resuspended to a final volume of 250 ml in buffer A and stored at -80 "C. Approximately 2400 mg of microsomal protein was obtained from 60 g wet weight of cerebellum.

r3H1IP3, PHIIP,, and r3H]IP6 Binding Assays [3H]IP4, [3H]IP3, and ['H]IP, binding (specific activity = 17, 17,

and 12 Ci/mmol, respectively) were used to monitor purification of the receptor. Binding of [3H]IP3 and [3H]IP4 was performed as de- scribed previously (5) except that, in order to conserve ligands, nonspecific binding was determined in the presence of heparin (100 pg/ml) which gave similar values to that determined in the presence of excess IP3, IPa, and IPS.

For competition studies of [3H]IP4 binding to purified receptor, 0.2 or 1.0 pg of receptor protein was assayed in 100 pl of binding medium composed of 25 mM Na-Hepes pH 7.1 (pH 7.4 on ice), 1 mM EDTA, 1 mM DTT, 1% CHAPS, 5 mg/ml equine IgG (Sigma), 10 nM [3H]IP4 (17 Ci/mmol) in the presence of 0-3.1 p~ unlabeled ligand and +lo0 mM KCl. The receptor was coprecipitated with IgG using 6% (w/w) polyethylene glycol and pelleted by centrifugation as de- scribed previously (5). Nonspecific counts were determined by omis- sion of the purified receptor from the binding medium. Competition curves were generated by fitting the data to a generalized ligand binding equation: y = a / [ l + (n/b)'] by nonlinear regression (Mar- quart-Levenberg algorithm), where a is maximal binding (normalized to loo%), b is the and c is the slope factor where a value of 1 indicates normal hyperbolic binding while values greater or less than one suggest either positive or negative cooperativity, respectively (see Ref. 29).

For Scatchard analyses, [3H]IP3, [3H]IP4, and [3H]IP6 stock solu- tions were either used as obtained from Du Pont-New England Nuclear (0.59, 0.59, and 0.80 p ~ , respectively) or were supplemented with "cold" IP3, IP,, and IP,, respectively, to give stock concentrations of 5.9 p M for IP3 and IP, (-2000-2200 cpm/pmol, -1.7 Ci/mmol), or 4.0 p~ for IPS (-2400 cpm/pmol, -2.4 Ci/mmol). The binding studies were then carried out as a function of ligand concentration by addition of increasing amounts of radioligand solution. The concentrations of the respective ligands used in all binding assays were calculated from information provided by the suppliers of radioactive and nonradioac- tive inositol phosphates.

Purification of the Inositol Polyphosphate Receptor Solubilization of the Microsomes-Frozen microsomes from 120 g

buffer B (50 mM Tris-C1 (pH 7.4), 1 mM EDTA, and 1 mM DTT). (wet weight) of cerebellum were thawed and mixed with 1500 ml of

Solubilization was initiated by addition of 1000 ml buffer B containing 60 g of CHAPS (final concentration = 2%, w/v). After gentle stirring for 1 h, the suspension was centrifuged for 1.5 h at 14,000 rpm using a Beckman JA-14 rotor. The pellets were discarded, and the super- natant was used for purification of the inositol polyphosphate recep- tor.

Purification of the Receptor-The supernatant was adjusted to 200 mM with NaCl and gently mixed with 85 ml of heparin-agarose (which had been equilibrated with H,O). After 2.5 h, the gel was collected by vacuum filtration, washed with 400 ml of buffer B containing 1% (w/ v) CHAPS and 200 mM NaCl, and poured into a 2.5-cm inner diameter

' The term microsomes is used in this study to refer to the pellet obtained from cerebellum homogenate by sedimentation at 19,000 X g., for 1 h.

column. The receptor was eluted with the same buffer containing 800 mM NaCl and 8-ml fractions were collected. The five peak fractions (Nos. 12-16) containing IP4 binding activity were pooled. The pooled fractions from the heparin-agarose column were dialyzed overnight against 1100 ml of buffer C (20 mM Tris-C1 (pH 7.4), 1 mM DTT, and 1 mM EDTA containing 1.0% (w/v) CHAPS). The dialyzate was diluted with an equal volume of buffer C and centrifuged for 30 min at 20,000 rpm using a Beckman Ti 70 rotor to remove aggregated protein. The supernatant was loaded onto a 15 ml column of DEAE- Sepharose-GB (1.5 cm inner diameter) at a flow rate of 0.5 ml/min, which had been prequilibrated with buffer C. Elution was achieved with a 150-ml gradient of 0-250 mM NaCl in buffer C, 7.5-ml fractions were collected. Peak fractions containing IP, binding activity (frac- tions 11-14) were pooled, frozen in liquid nitrogen, and stored at

After thawing, the pooled fractions were loaded at 0.5 ml/min onto a second heparin agarose column (3 ml, 1.0 cm inner diameter) which had been equilibrated in buffer D (50 mM Tris-C1 (pH 8.31, 1 mM EDTA, 1 mM DTT) containing 1.0% CHAPS. Elution of the receptor was achieved with a 40-ml gradient of 0.2-0.8 M NaCl in the same buffer, and fractions of 2 ml were collected. Fractions 8-15 containing the receptor were pooled and adjusted to 2.5 M NaC1. The sample was then applied onto a 2.0-ml column of phenyl-Sepharose 4B which had been equilibrated with buffer D containing 2 M NaCl and 1.0% CHAPS. The column was washed with 6 ml of the same buffer containing 2 M NaCl and the receptor obtained by sequential elution with 4 ml each of 1, 2,3, and 4% (w/v) CHAPS in buffer D (see Fig. 3B). Fractions (2 ml) containing IP4 binding activity, which eluted at CHAPS concentrations of 2% or more, were pooled. The pooled fractions from the phenyl-Sepharose column were concentrated (Cen- tricon-30) to 1-2 ml and loaded onto a TSK gel G3000SW column (60 cm X 21 mm) and eluted at 1.5 ml/min with buffer E (50 mM imidazole (pH 6.9), 100 mM NaC1, 1% CHAPS, 1 mM EDTA, 1 mM DTT). Fractions of 2.4 ml were collected and elution was monitored by AZm. Peak fractions (43-46) which contained the IP4 binding activity, were pooled, concentrated (Centricon C-30), and stored frozen at -80 "C. In practice, a single preparation was performed over a period of five days.

-80 "C.

Reconstitution into Planar Bilayers and Channel Measurements Preparation of Lipidlprotein and Lipid Vesicle-The lipid used

throughout this study was a lipid mixture: soybean lipid (Sigma, type 11-S, acetone-washed) with cholesterol (Fluka) in a weight ratio of 24:l. Lipid vesicles and lipid/protein vesicles were prepared in either 100 mM KC1 or 100 mM NaCl buffered to pH = 7.4 by 10 mM Hepes- Tris. Adjustments to other salt concentrations used, i.e. 500 mM KC1 or additional CaC12, were done after bilayer formation. Lipid vesicles were prepared by resuspending a thin film of 10 mg of lipid dried under nitrogen in 10 ml of buffer (15). Protein/lipid vesicles prepa- ration: 10 pl of receptor protein (0.54 mg/ml), 6 mg/ml CHAPS, and 3 mg/ml soybean lipid were suspended in 1 ml of buffer for 10 min with occasional vortexing. Then Bio-Beads (Bio-Rad) were added (0.23 g/ml), and the flask rotated for 1 h, followed by a second 1-h rotation with fresh 0.33 g/ml Bio-Beads. The sample was stored on ice until use (up to 3 h).

Planar Lipidlprotein Bilayers Formation-Planar bilayers were formed from vesicles of defined lipid protein ratio, according to the septum-supported vesicle-derived bilayer technique (15). Briefly, planar bilayers (aperture size of 0.18 mm) are formed by apposition of two vesicle derived monolayers. Both cis (protein-containing side) and trans chambers contain 1 ml of solution. Five (Figs. 10 and 12) and 2.5 pl (Fig. 11) of fresh protein lipid vesicle preparation was diluted into 2 ml of fresh lipid vesicle preparation to provide a working suspension of proteolipid vesicles. Using the molecular weights, 400,000 for IP, receptor and 750 for soybean phospholipid, the two above protein concentrations correspond to final molar ratios of protein/lipid in vesicles of 2.2 and 1.1. lo-', respectively, as given in the figure legends.

IP4 (Boehringer Mannheim) was applied in 1-pl aliquots directly to the membrane on the cis side via a steel tube. The tube, of inner diameter 0.85 mm, was adjusted with its end to the membrane (center to center) a t a distance of 0.15 mm. It could be removed for refilling and accurately replaced to the same position. Using valinomycin (concentration of 0.66 ng/ml, 1 p1 applied) for calibration, the time for half-maximal response of induced current was 6 + 1 s. The response showed a plateau between 30 s and 3 min followed by a slow decay (50% after 12 k 1 min) due to diffusion loss of the applied

Purification of an Inositol Polyphosphate Receptor 3475 valinomycin into the bulk solution (10).

Electrical Measurements and Data Processing-Electrical contact with the solution was made via Ag/AgCl electrodes. Voltage is ex- pressed as the voltage applied to the cis solution. The voltage signal across the feedback resistance (10'' ohms) of the current-measuring operational amplifier was filtered at 3 kHz and stored on a pulse code-modulated audio tape recorder modified to accept dc signals.

RESULTS

Purification and Characterization of the Inositol Polyphos- phate Receptor-Competition of [3H]Ins(1,3,4,5)P4 binding to bovine brain cerebellum microsomes at pH 8.9 detects low affinity Ins(1,3,4,5)P4 binding with a K d in the range of -1 pM (Fig. 1).

To further characterize this low affinity IP, receptor, its purification was achieved as described under "Experimental Procedures" and summarized in Table I. We have designated this receptor the inositol polyphosphate receptor since it also binds IPS and IPS (see below). Microsomes from cerebellum

1 0 - ~ IO-* 10" IO-^ 1 0 - ~

[Ins(l ,3,4,5)-P41

FIG. 1. ['H]IP4 binding to bovine cerebellum microsomes and competition by unlabeled IP4. [3H]IP4 binding was deter- mined with 0.1 mg of cerebellum microsomal protein in 0.1 ml of binding medium composed of 50 mM Tris-C1 (pH 8.9), 1 mM EDTA, 1 mM DTT, 10 nM [3H]IP4 (17 Ci/mmol), and the indicated amounts of unlabeled IP,. Nonspecific binding, determined in the presence of 100 pg/ml heparin, accounted for 2% binding, whereas total binding was 5% of the total radiolabel. An IC60 value of 0.83 p~ for unlabeled IP, indicates the presence of low affinity IP, binding in these micro- somes. The curve was generated by fitting the data to the equation y = a/ l + (n/b)' by nonlinear regression (Marquardt-Levenberg algo- rithm) (see "Experimental Procedures").

TABLE I Summary of receptor purification and IP4 binding

The results presented are for one of five typical preparations.

Fraction

mg Solubilized microsomes 4827 1623 0.34 Supernatant 1177 1302 1.11 First heparin column pool 152 902 5.9 DEAE-Sepharose poolb 19.4 76 3.9 Second heparin column pool 8.0 47 5.9 Phenyl-Sepharose pool 2.2 37.4 17.0 TSK pel G3000SW uool 0.36 16.7 46.4

-fold %l

1 100 3.3 80

17.4 56 11.5 4.7 17.4 2.9 50.0 2.3

136.5 1.0 "The concentration of [3H]IP4 used to determine binding was 10

nM. Binding of [3H]IP, was found to be inhibited by increasing concentrations of NaCl (C. C. Chadwick and S. Fleischer, unpublished results). Therefore, the concentration of NaCl in eash assay was adjusted to 40 mM.

* Approximately 50% of the [3H]IP4 binding was lost in the precip- itate obtained after dialysis of the pooled fractions from the first heparin column, accounting for a major loss of IP4 binding prior to the DEAE-Sepharose step.

were solubilized with CHAPS, and the receptor was purified using a combination of column chromatography procedures. Approximately 80% of IP, binding activity was solubilized by extraction of cerebellum microsomes with 2% CHAPS. The [3H]IP4 binding activity was then concentrated and enriched by absorption on heparin-agarose and elution with buffer containing high salt (Table I). After dialysis to lower the NaCl concentration, the IP, binding activity was purified by se- quential chromatography on DEAE-Sepharose 6B, a second heparin-agarose column, and phenyl-Sepharose 4B. The final step made use of a TSK gel G3000SW gel filtration column. The receptor is enriched approximately 140-fold from the solubilized microsomes, with an estimated recovery of 1%. A significant loss of IP, binding equivalents is sustained prior to the DEAE-Sepharose 6B step (Table I). This is attributable in part to co-precipitation of IP, binding activity (-50%) together with contaminating proteins during the dialysis step preceding the DEAE-Sepharose 6B chromatography.

The protein profile of the fractions obtained in the purifi- cation procedure was characterized by SDS-PAGE (Fig. 2). The purified receptor consists of three polypeptides with apparent molecular weights of 111,000 & 1.0 (mean & S.D., n = 3), 102 f 1.1 (mean & S.D., n = 3), and 52.0 f 1.0 (mean f S.D., n = 3). In the enriched fractions of the receptor from the phenyl-Sepharose (Fig. 3B, fractions 7-9) and TSK Gel gel filtration columns (Fig. 4B, fractions 43-45), a constant ratio of the three polypeptide bands is observed, as determined by densitometry, which correlates with [3H]Ins(1,3,4,5)P4 binding. These findings suggest that the receptor is a heter- ooligomeric complex of the three bands. Pretreatment of the purified receptor with 2-mercaptoethanol and heating (100 "C, 1 min) did not change the electrophoretic profile (not shown).

The three bands characteristic of the receptor are co-en- riched at each step in the purification (Fig. 2, lanes 2-6). The enrichment, observed in each of 10 preparations, correlates with the determinations of IP, binding activity (Table I), with the exception that there is a decrease in specific binding in the DEAE-Sepharose 6B pool compared with the pooled

200.0-

116.3- 97.4-

66.2-

45.0.

-DF 1 2 3 4 5 6

FIG. 2. Protein profile of fractions obtained in the purifi- cation of the inositol polyphosphate receptor. SDS-PAGE was carried out on a 7.5% polyacrylamide gel using the buffer system of Laemmli (24). Samples were treated with 1% 2-mercaptoethanol, 2% SDS at room temperature (5 min) prior to electrophoresis. The gel was stained with Coomassie Blue R-250. Lane 1, supernatant extract of cerebellum microsomes with 2% CHAPS; lane 2, pooled fractions from the first heparin-agarose column; lane 3, pooled fractions ob- tained from the DEAE-Sepharose 6B column; lane 4, pooled fractions obtained from the second heparin-agarose column; lane 5, pooled fractions obtained from the phenyl-Sepharose 4B column; lane 6, pooled and concentrated fractions from the TSK gel G3000SW col- umn. The protein loaded onto each lane was 1.25 pg as determined by the Kaplan and Pedersen method (16). The numbers on the left of the gel indicate the positions of M, standards: myosin, M, = 200,000; &galactosidase, M, = 116,250; phosphorylase b, M, = 97,400; bovine serum albumin, M, = 66,200; ovalbumin, M, = 45,000. The three bands referable to the inositol polyphosphate receptor are indicated by arrowheads.

3476 Purification of an Inositol Polyphsphute Receptor

A FT 5 6 7 8 9 1 0 1 1 1 2

Phenyl Sepharose Elution Profile E

1 4 , , , , , , , 12 1% 39: 4% CHAPS

Fraction FIG. 3. Purification of the inositol polyphosphate receptor

using phenyl-Sepharose chromatography. Fractions, from the second heparin column enriched in IP. binding, were applied directly to a column of phenyl-Sepharose (2 ml) and eluted by increasing the CHAPS concentrations as indicated in B (see “Experimental Proce- dures”). Each fraction collected was 2.0 ml. A, SDS-PAGE character- ization of column fractions. The buffer system of Laemmli was used on a 7.5% separation gel. The samples applied (12 pl) were: A, applied starting material, FT, flow-through showing unbound proteins, 5-12 are column fractions. B, correlation of IP, (0) and IP3 (W) binding at 10 nM [”]ligand with the Coomassie Blue relative absorbance of the three characteristic bands of the receptor a t 111 (0), 102 (A), and 52 (0) kDa.

fractions from the first heparin-agarose column. The molar ratio of 111-, 102-, and 52.0-kDa subunits, esti-

mated by densitometric scanning of Coomassie Blue-stained SDS-PAGE gels of four purified receptor preparations is 1.0:3.4 f 0.82.4 f 0.5 (n = 4 for each band).

Molecular sieve HPLC reveals a molecular weight of -400,000 for the purified receptor in 1% CHAPS (Fig. 5). Negative staining electron microscopy reveals uniform glob- ular-like structures of approximately 10-13 nm in diameter (Fig. 6).

Characterization of IPc Ips, and IPS Binding to the Purified Receptor-Binding of [3H]Ins(1,3,4,5)P4 to the purified recep- tor is not unique, since competition is observed not only with IP4, but also with IPS (Fig. 7A) and IP3 (Fig. 7B). The binding by IP4 and IPS at pH 7.4 appears to be high affinity but is attenuated by ionic strength. In a buffer of 25 mM Na-Hepes (pH 7.4), Ins(1,3,4,5)IP4, and InsP6 similarly compete for binding with ICm values of -25-35 nM. However, binding is weaker when measured under conditions approximating more physiological ionic strength (100 mM KC1, 25 mM sodium- Hepes (pH 7.4)). The estimated ICso values for both Ins(1,3,4,5)P4 and InsP6 are substantially increased (>5-fold) to 150 nM (Fig. 7A and Table 11). Ins(1,4,5)P3 also inhibits [3H]Ins(1,3,4,5)P4 binding (Fig. 7B). The estimated ICm value for Ins(1,4,5)P3 increases about 2.5-fold in going from the lower ionic strength to physiological ionic strength at pH 7.4. The relatively higher ICso, values under conditions approxi- mating physiological pH and ionic strength, indicate that in situ the receptor has weaker affinity for these inositol poly- phosphate ligands.

A

200 :

116.3 :

97.4 :

66.2 :

42.7 :

2 , A 42 43 44 45 46 47

41 42 43 44 45 46 47 48

Fraction FIG. 4. Purification of the inositol polyphosphate receptor

using HPLC gel permeation chromatography. Fractions from the phenyl-Sepharose column enriched in ligand binding were pooled, concentrated (2 ml), and applied to a TSK-gel G3000-SW column (60 cm x 21.5 mm inner diameter). Elution was at 1.5 ml/min, and 2.4- ml fractions were collected (see “Experimental Procedures.”) A, SDS- PAGE protein profile of applied sample ( A ) to and fractions eluted from gel filtration column (12 pl each). A, sample applied; fractions 4248; M, molecular weight markers. Molecular weight markers (M) are: myosin (200,000); galactosidase (116,300); phosphorylase B (97,400); bovine serum albumin (66,200); and ovalbumin (42,700). B, correlation of IP3 (W) and IP, (0) binding at 10 nM ligands with Coomassie Blue absorbance of the three bands of the receptor, 111 kDa (O), 102 kDa (A), and 52 kDa (0).

Binding parameters of the purified receptor were also ob- tained from Scatchard analyses (Fig. 8) for the three inositol polyphosphates, at conditions used to monitor purification of the receptor, i.e. pH 8.9 and 55 mM salt. Binding is character- ized by a Kd of 0.64 pM for Ins(1,3,4,5)P4, Kd = 0.49 pM for Ins(1,4,5)P3, and Kd = 0.12 p~ for InsP6 (Table 111). The Kd for Ins(1,3,4,5)P4 binding to the purified receptor (0.64 p ~ ) is in the same range as that estimated for Ins(1,3,4,5)P4 binding to the starting microsomes (-0.8 p ~ , Fig. 1) when measured at comparable conditions. Thus, the binding affinity with regard to Ins(1,3,4,5)P4 binding is essentially unchanged by purification.

Binding of [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,4,5)P3 at pH 8.9 and 55 mM salt are inhibited by ATP (Ki -100 p ~ ) , GTP (Ki -100 p ~ ) , and heparin (Ki -1 pg/ml); the Ki values are about the same for both ligands. Ins(1)P and Ins(l,4)Pn do not effectively compete for either Ins(1,4,5)P3 or Ins(1,3,4,5)P4 binding (Ki >lo0 p~ for both).

The finding that the receptor also binds IP, and IP, was unexpected. The previously isolated IP3 receptor from cere- bellum is characterized by a polypeptide with a M, of 260,000 as determined by SDS-PAGE which binds IP3 with a Kd in the nanomolar concentration range (Refs. 3-6). Scatchard analysis of IP3 binding to cerebellum microsomes is shown in

Purification of an Inositol Polyphosphate Receptor 3477

Time (min)

FIG. 5. Molecular weight determination of inositol poly- phosphate receptor by gel filtration HPLC. Molecular weight estimate of the purified inositol polyphosphate receptor by gel filtra- tion HPLC. The purified receptor (25 pg of protein) was applied in a volume of 0.10 ml to a TSK gel G4000SWXL column (0.75 x 30 cm) pre-equilibrated in 20 mM imidazole CI (pH 6.8), 250 mM NaCI, 2 mM DTT, and 1% CHAPS. The flow rate was 1.0 ml/min and absorbance was monitored a t 280 nm. M, standards which were chromatographed in the same buffer without CHAPS included thy- roglobulin, 669,000; ferritin, 440,000; and catalase, 232,000. The elu- tion position of standards is indicated by the closed circles. The elution profile of the receptor is shown in the figure. An M. of 400,000 was estimated for the receptor.

FIG. 6. Electron microscopy of inositol polyphosphate receptor. Electron micrograph of the purified receptor obtained after negative staining with uranyl acetate. The receptor has a diameter of 10-13 nm.

Fig. 9. The Scatchard plot is consistent with a t least two classes of binding sites. The higher affinity site is likely referable to the high affinity IP3 receptor (3-6), whereas the lower affinity site corresponds, at least in part, to the receptor described in this study. Curve fitting of the binding data was performed to estimate the relative density of the two binding sites (Fig. 9, cf. legend). The results indicate that the higher affinity site (& = 2.1 nM) has a B,,, of -0.83 pmol/mg, whereas the lower affinity site ( K d -1.4 p M ) has a Bmax of -19 pmol/mg. Thus, in cerebellum microsomes, we estimate the magnitude of the lower affinity binding site to be about an order of magnitude greater than the higher affinity binding site.

When the microsomes are extracted with CHAPS as de- scribed under “Experimental Procedures,” only a relatively small fraction, approximately 10% of the total IP3 binding, is referable to the high affinity IP3 receptor as monitored by its sedimentation into the lower regions of sucrose density gra- dients (5). The high affinity Ins(1,4,5)P3 receptor is not eluted from the DEAE-Sepharose 6B column under the conditions used to elute the inositol polyphosphate receptor.

Channel Characteristics of the Purified Inositol Polyphos- phate Receptor-The purified receptor preparation was recon- stituted into lipid vesicles from which planar membranes were formed (see “Experimental Procedures”). Ion channel activi-

1 0 - ~ IO-^ 1 0 - ~ 1 0 - ~ [Ins(l ,3,4,5)-P4] or [IP6]

0- 1 0 - ~ IO-^ 1 0 - 7 10-6 10-5

[Ins(l ,4,5)-P31

FIG. 7. Competition of [‘H]IP4 binding by IP,, Ips, and IPe at neutral pH (7.4). A, IP, (0) and IPS (A) at low salt (25.0 mM NaHepes); IP, (0) and IPS (A) a t “isotonic salt” concentration (25 mM NaHepes + 100 mM KCI). B, IP3 a t low salt (W) and isotonic salt (0). [‘HIIP, (10 nM) was supplemented with cold ligand as indicated in the figure. Data from two such studies are summarized in Table 11. The percentage of bound [3H]IP4 relative to total radiolabel ranged from 10 (at low ionic strength) to 3% (in isotonic salt). Nonspecific binding varied from 3% (low ionic strength) to less than 1% (isotonic salt).

TABLE I1 Competition of PHJIP, binding to the purified receptor with IPS, IP4,

and IPS Binding assays, determination of IC, values and slope factors were

carried out as shown in Fig. 7, A and B (see “Experimental Proce- dures”), on two receptor preparations. The binding assay was per- formed a t pH 7.4 in a medium containing low salt (25 mM NaHEPES, pH 7.4) or supplemented with 100 mM KC1 (“isotonic salt”). The values in parenthesis represent the range of IC, values and slope factors determined for each preparation. A slope factor of 1.0 indicates simple hyperbolic binding; a slope factor of less than one indicates negative cooperativity (29).

Low salt Ligand

Isotonic salt

IC, Slope factor ICso Slope factor

IrM IrM

IPS 0.23 0.64 0.58 0.65 (0.20-0.26) (0.57-0.70) (0.43-0.76) (0.58-0.72)

(0.025-0.038) (0.74-0.92) (0.16-0.22) (0.68-0.94) IPI 0.032 0.82 0.19 0.81

lpS 0.024 0.85 0.14 1.1 (0.014-0.033) (0.72-0.98) (0.10-0.17) (0.86-1.30)

ties were clearly observed in 45 out of 60 membranes and were dependent on the presence of the receptor, in contrast to lipid membrane controls which showed no channel activity a t all. Fig. 10 shows bursts of channel events of about 10-s duration which are typical for a KCl/NaCl gradient (100/100 mM) with positive potential applied to the cis side containing KC1 (50 mV in Fig. 10). The upper burst was observed just

3478 Purification of an Inositol Polyphosphate Receptor

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Bound (nmole/mg)

FIG. 8. Scatchard analysis of IPa m), IPS (A), and IPS (0) binding to the purified IPa/IPs receptor. Specific (duplicate samples) and nonspecific binding were determined at pH 8.9 in 50 mM Tris-C1 and 5 mM NaCl using -1.25 pg of purified receptor from 18 to 590 nM for [3H]IP4 and [3H]IP3 (2200 cpm/pmol) or 12.5-400 nM for [3H]IP6 binding (2300 cpm/pmol). Representative plots are shown. Correlation coefficients ranged from 0.99 to 0.94 for analyses of IP, binding, 0.97 to 0.99 for IPS binding, and 0.99 to 0.95 for IP3 binding. The percentage of bound ligand relative to the total radioli- gand ranged from 3.8 to 8% for [3H]IP4, 2 to 6.8% for [3H]IP3, and 4.5 to 15% for IPS. Nonspecific binding for all ligands was 2% of the total. Binding parameters from three or more experiments with three different preparations and assay conditions are summarized in Table 111.

TABLE 111 Scatchard analysis of binding data to the purified receptor

Binding was carried out at pH 8.9 in a medium containing 50 mM Tris-C1 and 5 mM NaCl, see Fig. 8. For IP, and Ips, nonspecific binding was equivalent to -2% total ligand. For IPS, -3% of total ligand. IP3 and IP, concentrations ranged from 18 to 590 nM. IPS concentrations ranged from 12 to 400 nM. Specific activities: IPS, 2200 cpm/pmol; IP4, 2200 cpm/pmok IPS, 2400 cpm/pmol.

Ligand K d B , Correlation coefficient

CM nmollmg IP3 0.49 f 0.17 1.86 f 0.31 6 0.94-0.99 IP, 0.64 f 0.21 3.0 f 0.33 5 0.94-0.99 IPS 0.12 f 0.03 1.29 f 0.67 3 0.95-0.99

after bilayer formation and the middle burst 30 min later with 1 2 bursts of similar duration in the intermediate time. These types of bursts were observed in three out of three membranes studied at conditions of Fig. 10. Occasionally, a second class of bursts appeared with a much higher transition frequency as shown in the lower trace of Fig. 10. The mean open time of the channel was strongly dependent on ionic conditions. At symmetrical KC1 (100 mM), channel open times were much longer, in the 1-10-min time range (cf. lowest trace in Fig. 11). Under these conditions, the channel showed a constant con- ductance of 7.2 f 0.8 pS in the range of -100 to +lo0 mV applied potential and no significant dependence of mean open time with applied voltage (data not shown).

IP4 was found to close the channel. The long mean open time of the channel in symmetrical 100 mM KC1 was used to study effects of inositol polyphosphates on this channel activ- ity. A key finding is illustrated in Fig. 11. It shows a contin- uous record where channel activity was completely blocked by 10 p~ IP4 (Ins(1,3,4,5)IP4 when applied close to the mem- brane (cis side with 1 pl of 10 PM IP4, using a precisely readjustable tube, see "Experimental Procedures")). Upon tube removal and 30-s stirring (dilution of IP4 to -10 nM), channel activity reappeared. Channel blockage by 10 p~ IP4 and reopening upon IP4 dilution was repeated three times;

0.06 1, \ 1

0.05

v E 0.04 1 1 $ 0.03

0 4 8 12 16 20

Bound (pmole/mg)

FIG. 9. Scatchard analysis of IPS binding to cerebellum mi- crosomes. Specific (duplicate samples) and nonspecific binding were determined using 0.12 mg of microsomal protein/incubation and from 25 to 590 nM [3H]IP3 (2200 cpm/pmol). Specific binding ranged from 5.2 to 1.1% of total ligand, nonspecific binding was 2.0% of total ligand. Binding data was also analyzed by nonlinear regression. A best fit was obtained using a double rectangular hyperbola model (not shown), in which the K d of the higher affinity site was fixed at 2.1 nM (a value which we have previously determined, using identical conditions, for the purified high affinity IP3 receptor from brain (6)). Other binding parameters were determined by iteration. The esti- mated values were: (high affinity) = 0.83 k 0.21 pmol/mg, K d

(low affinity) = 1.4 k 0.6 pM, Bmax (low affinity) = 18.9 & 5.1 pmol/ mg. The correlation coefficient was 0.986.

FIG. 10. Reconstitution of inositol polyphosphate receptor into planar bilayers. Channel events were measured at a KCl/ NaCl gradient. Holding potential was +50 mV (cis side). Aqueous phase conditions, cis side: 100 mM KCl, vesicles at a protein/phos- pholipid molar ratio of 2.2.10"'; trans side: 100 mM NaCl, lipid vesicles. Traces were filtered with 300 Hz.

two additional cycles are shown in the figure. The final trace (note the 3-fold compressed time scale) shows that, without application of 10 p~ IP4, the channel remained open for more than 15 min with occasional sojourns to the closed state.

Complete blockage by 10 p~ IP, was observed in four out of four planar bilayer membranes (Fig. 11). Application of 10 pM IP3 (Ins(1,4,5)P3) or 10 pM IPS at conditions of Fig. 11 did not activate additional channels or block the observed chan- nels. In the presence of 1 p~ IP, (conditions of Fig. 11) the long lasting open channel events were not completely blocked, but convert to flicker behavior, i.e. repetitive transitions be- tween open and closed states, albeit similarly long lasting trains of rapid closing and reopening events (1-10 min). Such activity is exemplified in Fig. 12a, where it has been employed to determine the permeability ratio PK+/PC)- of the channel. The data were obtained in the presence of 1 PM IP4 and a KC1 gradient (500/100 mM). Open channel currents of the traces in Fig. 12a are plotted against applied voltage (Fig. 12b), yielding an estimate for the reversal potential of -35 * 2 mV. Using the common constant field approximation (17), a permeability ratio, PK+/PCL of -19 is obtained, indicating a high selectivity of K+ ions against C1- ions. A second mem- brane at the same conditions yielded virtually the same

Purification of an Inositol Polyphosphate Receptor a

+ lOOmV

3479

s- p Y & , a! 90rec

-1 h r+-q qp FIG. 11. Blockage of single channel events by 10 pm IPS.

Spontaneous single channel activity was blocked by applying 1 pl of solution containing 10 PM IP, close to the cis side of the membrane (for technical details, see “Experimental Procedures” section). After dilution of IP, by stirring into the cis solution (1 ml), channel activity reappeared. This is repeated two times in the two middle traces, whereas the lowest trace confirms long channel open times without IP, application at a three times condensed time scale. Aqueous phase conditions were symmetrical with respect to KC1 (100 mM) and protein (molar protein/phospholipid ratio in vesicles was 1.1. lo-’). Traces were filtered with 100 Hz.

“flicker” activity at 1 p~ IP4 and the same reversal potential. For the channel currents observed in a KCl/NaCl gradient (100/100 mM) as shown in Fig. 10 for +50 mV applied to the KC1 side, a reversal potential of -35 to -40 mV was obtained. From this value and from the permeability ratio PK+/PcI- of -19 a value of PK+/PNa+ of -5 is obtained (17).

Attempts to determine a value for PK+/Pc~~+ failed thus far. High Ca2+ concentrations (above 5 mM) applied to either side of the membrane abolished channel activity observed before a t symmetrical KC1 (conditions of Fig. 11). Rare channel events (at 50 mM CaClz at the trans side) indicated, however, that the reversal potential remained at 0 -+ 5 mV, which sets Ca2+ permeability of the channel to rather low values. In addition, Ca2+ (50 mM) appeared to reduce channel currents for applied voltages of either sign by about a factor of 3, which also rendered determination of reversal potentials difficult (data not shown).

DISCUSSION

We have purified an inositol polyphosphate receptor from bovine cerebellum which binds IP,, IP3, and IPS. The receptor preparation, when reconstituted into planar bilayers, displays channel activity. IP, induces rapid channel flicker at 1 p~ concentration and closes the channel at 10 p ~ . IPS or IPS are without effect under similar conditions.

The receptor has been enriched about 140-fold in IP, bind- ing from the starting cerebellum microsomes (Table I) where it appears to exist at higher concentration by an order of magnitude than the IP3 receptor described previously (3-5) (Fig. 9). At conditions approximating physiological pH and ionic strength, the purified receptor has binding affinity (IC5o competition uers’sus [3H]IP4) in the range of -0.15, 0.2, and 0.5 pM for IPS, IP4, IP3, respectively. At lower ionic strength (25 mM), 5-10-fold higher affinity is observed for IP, and IPS and >2-fold for IP3 binding (Fig. 7 and Table 11). Heparin markedly blocks the binding of IP3, IP4, and IPS; inositol 1- phosphate and inositol 1,4-&phosphate do not effectively compete for binding of either IPS or IP,.

The purified receptor preparation consists of three bands

b

-0 4 1 FIG. 12. Current-voltage relationship. a, channel events at 1

p~ IP,, voltage-clamped on cis side from -70 mV to +lo0 mV. At 1 p~ IP, (final concentration on cis side), channel activity appeared reduced to consecutive brief openings. Opening rates and open times were not significantly dependent on voltage. IP, was applied to the cis solution from a 100 times concentrated stock solution before membrane formation. The traces shown were observed immediately after membrane formation. Cis solution contained 500 mM KC1 and trans solution 100 mM KC1. Other conditions as in Fig. 10. Traces were filtered with 100 Hz as the best compromise for resolving small current amplitudes close to current reversal (see the two lowest traces). b, current to voltage relationship from traces in a. Open channel currents were estimated from events with open times longer than 50 ms (at least 10 events at each voltage). Filled circles are larger than the standard error of the means. Connection of data points by the dashed line (hand drawn) yield a reversal potential of -35 k 2 mV.

with apparent molecular weights of 111,000, 102,000, and 52,000 as determined by SDS-PAGE (Fig. 2). The complex has an estimated stoichiometry of 1:3:2. This would be com- patible with the minimal molecular weight of about 500,000, assuming that each of the peptides had the same extinction coefficient with Coomassie Blue and that the electrophoretic mobility obtained by SDS-PAGE provides reliable estimates of molecular weight. Our data suggest specific association of the three polypeptides as a receptor complex: 1) the coenrich- ment of the 111,000, 102,000, and 52,000 bands as observed by SDS-PAGE (Figs. 2 and 3); 2) the symmetry of the HPLC gel titration elution profile (Figs. 4 and 5); and 3) the uniform size of the particles observed by electron microscopy (Fig. 6).

The Stokes radius of the purified receptor solubilized in CHAPS was determined by HPLC gel filtration to be

3480 Purification of a n Inositol Polyphosphate Receptor

-400,000 (Fig. 5). Negative staining electron microscopy re- veals a globular structure with a diameter of 10-13 nm (Fig. 6), consistent with a molecular weight in the range of 400,000. A receptor with M, of 400,000, which binds one mole equiva- lent of ligand, has an expected BmaX of 2.5 nmol/mg. This is in the range of B,,, values determined for IP,, but is somewhat lower for IPS and IP, (Table 111).

The reconstitution experiments and channel measurements provide evidence that the purified receptor preparation is associated with ion channel activity. Channel currents were strictly dependent on the presence of the receptor preparation. We find that IP, induces channel flickering in the concentra- tion range of the binding ( K d -1 p ~ ) , with complete blockage at 10 p M IP4 concentration. For initial classification of this channel, we studied permeability ratios of physiological ions (K+, C1-, Na+, Ca2+), which indicate that the channel may be classified as a potassium channel, although residual Ca2+ permeability cannot be excluded, at present. The localization of the receptor in the cell remains to be determined. The K+ permeability and sensitivity of its open time to asymmetric Na+ and K+ across the membrane might indicate that the channel is of plasma membrane origin. In this first study, the channel characterization was limited to show that the receptor preparation forms an ion channel after reconstitution into planar bilayers. Although inactivation of the channel by IP, is reported here, the nature of activation of the channel, i.e. what turns it on and why it is selectively modulated by IP,, remains to be discerned.

IP3 has been implicated in intracellular Ca2+ mobilization in a diversity of cell types and has been shown to release Ca2+ from microsomes derived from cerebellum (18-20), smooth muscle (21), and platelets (22). A high affinity IPS receptor (Kd = 2-80 nM) has been isolated previously from both cerebellum (3, 4) and smooth muscle ( 5 ) . The cerebellum IPS receptor has been cloned and shown to have a protomer molecular weight of 313,000 (4,6, 25). The smooth muscle IPS receptor is structurally and functionally similar to the cere- bellum receptor (6). The cerebellum IP3 receptor has been implicated as a Ca2+ channel, since IP3 enhances Ca2+ efflux from vesicles containing the reconstituted receptor (7, 8). More recently, we have reconstituted the purified IPS receptor from smooth muscle into planar lipid bilayers and have di- rectly demonstrated it to be an IP3 activated Ca2+ channel (10). Thus, the high affinity IP, receptor from cerebellum (3, 4) and smooth muscle ( 5 ) is an IP3-activated channel respon- sible for mediating release of intracellular Ca2+.

There is evidence that Ins(1,3,4,5)P4 may also play a role in the mobilization of intracellular Ca2+. IP4 appears to aug- ment IP3-dependent Ca2+ mobilization in mouse lacrimal aci- nar cells (12, 13). Ca2+ release from cerebellum microsomes has been reported to be triggered both by IP4 (23) and IP3 (19). IP4 has been reported to regulate K+ channels and intracellular Ca2+ in DDT, MF-2 smooth muscle cells (31).

The inositol polyphosphate receptor preparation described in this study is clearly different from the high affinity IPS receptors isolated previously from cerebellum (3, 4) and smooth muscle ( 5 ) . The high affinity IP, receptor is a tetramer of a single polypeptide chain which has a protomer molecular weight of 313,000 (4,6,25). Morphologically, the IP3 receptor exhibits 4-fold symmetry, appearing as a pinwheel with four arms radiating from a central hub (5). The inositol polyphos- phate receptor binds IP3 with lower affinity (-0.5 p~ uersus 110 nM) under comparable conditions (pH 8.9 and -50 mM salt) (this study and Ref. 5). The inositol polyphosphate receptor is a heterooligomer consisting of three polypeptides. I t is spherical and much smaller in size (10-13 nm, Fig. 5)

than the IP3 receptor which has dimensions of 25 X 25 x -10 nm (5). We estimate that in cerebellum, the inositol poly- phosphate receptor is capable of binding about 20-fold more IP3 than the high affinity IP, receptor (Fig. 9).

Significantly, IP3 kinase and IP, phosphatase activities (either Ca2+/calmodulin-dependent or -independent) is not detected in the purified inositol polyphosphate receptor, al- though readily detected in the first heparin pooled fraction.,

Values for intracellular IP3 concentration in the literature vary widely from submicromolar basal levels to several micro- molar or higher with activation (32). There is less data re- ported in terms of the IP, concentrations in tissues. Several micromolar of IP, or higher has been indicated which is higher concentration than the ICso for IP, binding (32).

Partial purification of a high affinity IP4 binding protein from cerebellum has previously been reported by Theibert et al. (26) and Donie et a1 (27). Both groups report binding specific for IP4 (& = 1-10 nM), but not for IP3. After our manuscript was submitted, the purification of a high affinity IP4 receptor and an IPS receptor was reported using an IP4 affinity column (30). The IPS receptor is a heterotrimer of similar subunit composition (115, 105, and 50 kDa) to that reported here for the inositol polyphosphate receptor and appears to have similar binding characteristics. Competitive binding studies using [,H]IP4 indicate that a number of ino- sitol polyphosphates (IP,, IP,, IPS, and IPS) bind to the receptor. The reported binding of IPS is high affinity (ICS0 -12 nM) at low ionic strength. We find that binding is considerably weaker at isotonic salt concentration and that binding of IP, and IPS are not very different.

We have designated the receptor the inositol polyphosphate receptor to reflect is promiscuity of binding. Reconstitution studies with the inositol polyphosphate receptor preparation indicate an IP4-modulated K+ channel. The role of the recep- tor and its further characterization, especially the channel gating and its relevance to function, remain to be determined.

Acknowledgments-We thank Dr. Lee Limbird, Department of Pharmacology, Vanderbilt University Medical School, for her advice on the binding studies and Laura Taylor for capable secretarial assistance.

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