Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells

Brain Research 1055 (2005) 60 – 72 www.elsevier.com/locate/brainres

Research Report

Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells James Watras a,*, Charles C. Fink c, Leslie M. Loew b a

Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032, USA b Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, USA c Department of Biological Sciences, University of Wisconsin, Milwaukee, WI 53201, USA Accepted 28 June 2005 Available online 10 August 2005

Abstract Cerebellar Purkinje neurons and neuroblastoma N1E-115 cells require 10 – 50 times more InsP3 to induce Ca2+ release than do a variety of non-neuronal cells (including astrocytes, hepatocytes, endothelial cells, or smooth muscle cells). Given the importance of InsP3-induced Ca2+ release for the development of synaptic plasticity in Purkinje neurons, a low InsP3 sensitivity may facilitate the integration of numerous synaptic inputs before initiating a change in synaptic strength. In the present study, attention is directed at the mechanism underlying this low InsP3 sensitivity of Ca2+ release. We show that permeabilization of neuroblastoma cells with saponin increased InsP3 sensitivity of Ca2+ release, indicating the presence of a diffusible, cytosolic inhibitor(s) of Ca2+ release. Consistent with this hypothesis, gel filtration of the neuroblastoma cytosol yielded three peaks that inhibited InsP3-induced Ca2+ release from permeabilized cells. The prominent inhibitory peak decreased the InsP3 sensitivity of Ca2+ release from permeabilized cells, did not bind 3H-InsP3, and was present in sufficient levels to account for the low InsP3 sensitivity of Ca2+ release in intact neuroblastoma cells. Purification of this prominent inhibitory fraction yielded a protein band that was identified by mass spectrometry as stress-induced phosphoprotein 1 (mSTI1). Furthermore, immunoprecipitation of mSTI1 decreased the inhibitory activity of N1E-115 cytosol, indicating that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release. Thus, the low InsP3 sensitivity of Ca2+ release in neuroblastoma cells can be explained by the presence of cytosolic inhibitors of Ca2+ release and include stress-induced phosphoprotein 1. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Second messengers and phosphorylation Keywords: Inositol 1,4,5-trisphosphate; Ca2+ channel; Cytosolic inhibitor; N1E-115 cell; Stress-induced phosphoprotein 1 (mSTI1)

1. Introduction Inositol 1,4,5-trisphosphate (InsP3) represents a critical second messenger for numerous hormones and neurotransmitters, elevating intracellular calcium (Ca2+) by activating an InsP3-gated Ca2+ channel in the endoplasmic reticulum. Differences, however, are apparent in terms of the sensitivity of various cells to InsP3. Studies using

* Corresponding author. Fax: +1 860 679 3125. E-mail address: [email protected] (J. Watras). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.091

photolytic uncaging of InsP3, for example, have shown that endothelial cells, hepatocytes, and astrocytes exhibit halfmaximal Ca2+ release upon photorelease of ¨0.2 AM InsP3 [24], whereas cerebellar Purkinje neurons require ¨25 AM InsP3 to initiate Ca2+ release [13,24]. Similarly, neuroblastoma N1E-115 cells [11] required 10– 30 times higher concentrations of InsP3 for half-maximal Ca2+ release than did the smooth muscle line A7r5 [10]. Regional differences have also been reported, with the soma of cortical pyramidal neurons [35] and neuroblastoma cells [11] exhibiting a higher InsP3 sensitivity than the distal dendrites. Given the importance of InsP3-induced Ca2+ release for the develop-

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ment of long-term depression in cerebellar Purkinje neurons [8,20,25], a low InsP3 sensitivity may provide a means for integrating synaptic inputs before initiating a change in synaptic strength. It remains to be determined if a low InsP3 sensitivity of Ca2+ release is a generalized property of neurons, though the reported difference in InsP3 sensitivity between soma and dendrites in pyramidal cells [35] and neuroblastoma cells [11] points to a low InsP3 sensitivity in confined regions such as the distal dendrites and spines, which would discourage a rise in intracellular Ca2+ at low levels of stimulation/neurotransmitter. The mechanism(s) underlying a low InsP3 sensitivity of Ca2+ release in neurons and neuroblastoma cells is not known, though it does not appear to be an intrinsic property of the InsP3 receptor. Instead, the low InsP3 sensitivity of some neurons has been hypothesized to be a consequence of a high density of InsP3 receptors [35], the presence of competitive inhibitors of InsP3 binding [17,19,31,42], and/or the presence of InsP3 binding molecules distinct from the InsP3 receptor [38]. In the present study, we used saponin permeabilization of neuroblastoma N1E-115 cells to release potential cytosolic inhibitors of InsP3-induced Ca release and show that saponin-permeabilized neuroblastoma N1E-115 cells exhibit a high InsP3 sensitivity. The latter result is consistent with the presence of a diffusible cytosolic inhibitor of InsP3-induced Ca2+ release in N1E-115 cells. The cytosolic fraction from the N1E-115 cells was then subjected to column chromatography, yielding three inhibitors of InsP3-induced Ca2+ release, which we calculate can explain the low InsP3 sensitivity of Ca2+ release in intact N1E-115 cells and implicate stressinduced phosphoprotein 1 in the inhibition of InsP3induced Ca2+ release.

2. Materials and methods 2.1. InsP3-induced Ca2+ release in intact cells InsP3-induced Ca release from intact N1E-115 cells and intact A7r5 cells was assessed using caged InsP3 techniques [10,11]. Specifically, N1E-115 cells were grown on coverslips in Dulbecco’s modified Eagle’s medium (DMEM; Gibco #11965) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (AA) for 4– 6 days (5% CO2, 37 -C). To induce differentiation of the N1E-115 cells (characterized by the development of axons and dendrites), the growth media was changed to DMEM supplemented with 0.5% FBS, 1% DMSO, and 1% AA for 2 days. The latter media (containing a low concentration of serum) has been shown to result in the development of axons and neurites in essentially all of the N1E-115 cells within 48 h [1,12,33], independent of protein synthesis [33]. Two days after the transfer of the N1E-115 cells to low serum media, the coverslip was mounted on the bottom of

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an open-welled plastic chamber and immersed in Earle’s Balanced Salt Solution (EBSS; pH 7.2). Cells were then micro-injected with a solution containing 1 mM NPE-InsP3 (Calbiochem), 0.667 mM calcium green – 1/10 kDa dextran (CG-1), 135 mM KCl, 10 mM aspartate, 10 mM HEPES (pH 7.2), incubated for 30 min at 37 -C, then calcium green fluorescence was monitored on an inverted confocal microscope (488 nm excitation laser line, 520 nm emission; 37 -C). Uncaging of the NPE-InsP3 was accomplished by 10 – 500 ms exposures to ultraviolet light from a mercury arc lamp (filtered <440 nM). The cytosolic concentrations of InsP3 were calculated from estimates of the NPE concentration in the cell and the efficiency of uncaging at a given exposure time, as described previously [9,11,32]. A similar procedure was used for analysis of the InsP3 dependence of Ca2+ release from intact A7r5 cells, except that A7r5 cells were grown in DMEM (Gibco #11995)/10% FBS/1% AA before being transferred to EBSS and then micro-injected with the above caged InsP3 solution. 2.2. InsP3-induced Ca2+ release in permeabilized cells N1E-115 cells were grown in 100 mm culture plates as described above, then after 2 days in differentiation media, the N1E-115 cells were displaced from the plates by a gentle stream of PBS, transferred to a 15 ml culture tube, and centrifuged 3 min at 30
g (22 -C). The pellet was gently resuspended in buffer A (20 mM HEPES, 120 mM KCl, 2 mM ATP, 12.5 AM EGTA, 0.5 mM MgCl2, pH 7, 22 -C) supplemented with 100 AM EGTA and then centrifuged as above. The pellet was gently mixed with buffer A containing 0.1% saponin and 112.5 AM EGTA, incubated 5 min at 37 -C, then centrifuged (30
g, 3 min, 22 -C). The saponin-permeabilized cells were resuspended in buffer A containing 15% sucrose and 112.5 AM EGTA (22 -C). For analysis of InsP3-induced Ca2+ release, aliquots (10 Al) of the saponin-permeabilized cells were allowed to accumulate 45Ca (in buffer A containing 5 mM MgCl2, trace 45 CaCl2, ¨200 nM free Ca2+) for 1 –5 min at 22 -C (as specified), then buffer (or a given column fraction or heparin) was added, followed 5 s later by addition of InsP3 buffer. Within 5 s after addition of InsP3 buffer, the mixture (130 Al total volume) was filtered (HSI filtration unit; 20 in. Hg vacuum; Millipore HAWP filters; 0.45 Am pore size; 25 mm diameter), then the filters were washed twice with 1 ml buffer B (20 mM HEPES, 120 mM KCl, 5 mM MgCl2, 1 mM EGTA, pH 7, 22 -C). Filters were assayed for radioactivity by liquid scintillation counting, with InsP3sensitive Ca2+ release representing the difference in radioactivity between samples treated with or without heparin. Comparable results were obtained if InsP3-sensitive Ca2+ release was calculated as the difference in radioactivity between samples treated with or without InsP3 in the release media, indicating that all of the InsP3-induced Ca2+ release measured under these assay conditions was heparin-inhib-

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itable. The InsP3 buffer used in all assays contained buffer A, with specified InsP3 concentration. Free Ca2+ concentration at the time of Ca2+ release was estimated to be 133 nM (based on fura-2 measurements). Total releasable 45Ca was determined by including ionomycin in the InsP3 release media for one sample. All Ca2+ release assays were completed within 40 min after saponin permeabilization. For analysis of InsP3-induced Ca2+ release from permeabilized A7r5 cells (a smooth muscle cell line), the A7r5 cells were removed from the culture plates by trypsinization (0.25% trypsin, 1 mM EDTA), diluted in 10 volumes of the growth media, centrifuged 3 min at 30
g (22 -C), and then washed in PBS. The A7r5 cells were washed again in buffer A, permeabilized with saponin, and assayed for InsP3induced Ca2+ release, as described for the N1E-115 neuroblastoma cells.

2.4. Immunoprecipitation N1E-115 cytosol was subjected to superpose 12 gel filtration and fractions assayed for inhibition of InsP3induced Ca2+ release from permeabilized A7r5 cells (as described above), yielding a prominent inhibitory peak at ¨400 kDa. Aliquots from the ¨400 kDa inhibitory peak were incubated/rotated with anti-mSTI1 (Stressgen; # SRA1500), anti-Hsp72/73 (Calbiochem; #HSP01), gel filtration buffer, or phosphate-buffered saline (used in the antibody solutions) for 4 h at 4 -C. An aliquot of prewashed proteinG Sepharose beads (Amersham Biosciences; #17-0885-01) was added to each vial, rotated for 45 min (4 -C), then centrifuged for 5 s at 16 k
g. An aliquot of the supernatant was then assayed for ability to inhibit InsP3-induced Ca2+ release from permeabilized A7r5 cells (as described above, using 0.7 AM InsP3).

2.3. Cytosolic extracts To assess the presence of cytosolic inhibitors of InsP3induced Ca2+ release in differentiated N1E-115 cells, a cytosolic extract was subjected to column chromatography, with eluates screened for inhibition of InsP3-induced Ca2+ release from permeabilized cells. Specifically, differentiated N1E-115 cells were washed in PBS, resuspended in 10 mM imidazole (pH 7; 2 -C; with 20 AM pepstatin A, 20 AM leupeptin, 20 AM aprotinin), and then sonicated (5 s, 3
, in ice bath). The sonicate was brought to 0.15 M KCl, centrifuged (20 min at 15 k
g (4 -C), 30 min at 100 k
g (4 -C)), and then subjected to gel filtration (superdex 30 resin; HR 10x30 column; equilibrated with 20 mM TRIS, 100 mM KCl, pH 7.6, 4 -C; 0.3 ml/min; exclusion limit 30 kDa). Eluates were assayed for ability to inhibit InsP3-induced Ca2+ release from permeabilized N1E115 cells and/or permeabilized A7r5 cells, as specified. Inhibitory fractions were further purified by superose 12 gel filtration (HR 10
30; equilibrated with 20 mM TRIS, 100 mM KCl, pH 7.6; 4 -C; 0.6 ml/min; exclusion limit 600 kDa), MonoQ anion exchange (HR 1
5; 1 ml/min; equilibrated with 20 mM TRIS, 20 mM NaCl, pH 7.6; eluting with a 0.02– 0.5 M NaCl gradient), and then a second round of superose 12 gel filtration. The ability of fractions to inhibit InsP3-induced Ca 2+ release from permeabilized cells was assayed as above. The protein concentration in the sonicate and eluates was determined using the Bio-Rad Coomassie blue G-250 staining technique (595 nm), with bovine serum albumin used as a standard. Purity of inhibitory fractions was assessed by SDSpolyacrylamide gel electrophoresis (using Coomassie blue staining to detect abundant proteins followed by Bio-Rad silver staining to detect minor proteins), with Bio-Rad kaleidoscope standards used to estimate molecular weight. The identity of electrophoretic bands from the purified inhibitory fractions was determined by tandem mass spectrometry, following in-gel trypsin digestion, as described previously [15,16].

3. Western blot analyses Comparison of the levels of mSTI1 in A7r5 cells and N1E-115 cells was accomplished using Western blot techniques. Specifically, A7r5 cells and differentiated N1E115 cells were solubilized in SDS sample buffer, boiled for 5 min, then subjected to SDS-polyacrylamide gel electrophoresis [26]. The amount of protein applied to the lanes varied (viz., 77 Ag, 38.5 Ag, 19.25 Ag, 9.62 Ag) for both the A7r5 cells and N1E-115 cells so as to assess linearity of the mSTI1 signal on the Western blot. Bio-Rad kaleidoscope standards were included in adjacent lanes to assess molecular weight. Proteins were transferred from the gel to PVDF [41], then the PVDF was probed for the presence of mSTI1 using antibody to mSTI1 (Stressgen; # SRA-1500) and Pierce chemiluminescent Supersignal West Pico IgG detection kit. Chemiluminescent signals from the Western blots were detected in real time by a UVP BioImaging Systems Epichemi3 Darkroom, equipped with a Hamatsu camera and Labworks image acquisition and analysis software. Quantification of the chemiluminescent signals was done subsequently using the UVP Labworks software. 3.1. Curve fitting Analyses of the InsP3 dependence of Ca2+ release and the inhibition constants for the various eluates were determined using least squares analyses (in Microsoft Excel). Specifically, the InsP3 dependence of Ca2+ release was fit to the equation    R ¼ ðRmaxTS n Þ= S n þ ðK n Þ4 1 þ ð I=KiÞni ; where R is the extent of Ca2+ release at the specified concentration of InsP3 and inhibitory fraction, Rmax is the maximal extent of Ca2+ release in the absence of inhibitor, S is the InsP3 concentration, n is the Hill coefficient for InsP3-

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induced Ca2+ release in the absence of inhibitor, and K is the concentration of InsP3 that can elicit half-maximal Ca2+ release in the absence of inhibitor. The values Rmax, K, and n were obtained by curve fitting Ca2+ release data in the absence of inhibitor. The terms Ki and ni represent the inhibition constant and the Hill coefficient for the particular inhibitor and were determined by fitting the above equation to Ca2+ release data at various InsP3 and inhibitor concentrations. In all of these analyses, the inhibitor concentration (I) was calculated as the dilution (i.e., percentage) of the N1E-115 cytosol in the Ca2+ release assay, given our previous measurements of the intracellular volume of differentiated N1E-115 cells (viz., 3 Al intracellular volume per mg homogenate) [11] and the dilutions of the cytosol during the chromatography and Ca2+ release assay. Inhibitor concentrations in the Ca2+ release assays amounted to 3 – 10% of that expected in cytosol of intact N1E-115 cells. The constants Rmax, Km, n, Ki, and ni obtained above were then used to predict the InsP3 dependence of Ca2+ release at a cytosolic concentration of a given inhibitor (by setting the inhibitor concentration to 100%; i.e., undiluted). 3.2. 3H-InsP3 binding To assess the possibility that various cytosolic inhibitors decreased InsP3-induced Ca2+ release by binding InsP3, 3HInsP3 binding by the cytosolic inhibitors was examined. Media was equivalent to that used for analysis of InsP3induced Ca2+ release, with 100 nM 3H-InsP3 final concentration. Specifically, cell suspension media (containing buffer A, 100 AM EGTA, 15% sucrose, 30 Ag gammaglobulin) was mixed with Ca2+ uptake solution and column buffer (or inhibitory fraction) in the same proportions as in the Ca2+ release assay. Ca2+ release media containing 3HInsP3 was then added followed immediately by 0.5 vol 45% polyethylene glycol and centrifugation (5 min at 45 k
g). The supernatant was removed by aspiration, then the pellet was solubilized in 100 Al Soluene 350, and assayed for radioactivity by liquid scintillation counting. Binding reactions were conducted at both 22 -C (as in the Ca2+ release assays) and at 4 -C (to minimize possible hydrolysis of 3H-InsP3 by endogenous phosphatases). As an internal control, 3H-InsP3 binding by canine cerebellar microsomes was also assessed under these conditions, in the presence and absence of 0.3 mg/ml heparin (a competitive inhibitor of InsP3-induced Ca2+ release [14]).

4. Results 4.1. Intact differentiated neuroblastoma N1E-115 cells exhibit a low InsP3 sensitivity of Ca2+ release compared to smooth muscle A7r5 cells As shown in Fig. 1, intact differentiated N1E-115 neuroblastoma cells micro-injected with caged InsP3 require

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higher levels of photolysed InsP3 to induce Ca2+ release than do intact A7r5 smooth muscle cells. Panels (A) and (B) show the time course of Ca2+ release after uv-induced photolysis of InsP3, with N1E-115 cells requiring at least 0.3 AM InsP3 to initiate Ca2+ release, whereas Ca2+ release from A7r5 cells could be initiated after uv-induced photolysis of 0.01 AM InsP3. This difference in InsP3 sensitivity is further evidenced by Fig. 1C, which plots the percent maximal Ca2+ release as a function of InsP3 concentration, with intact neuroblastoma cells and intact A7r5 cells exhibiting halfmaximal Ca2+ release at 1.25 T 0.28 AM InsP3 and 0.10 T 0.02 AM InsP3, respectively (panel D). Hill coefficients for InsP3-induced Ca2+ release from intact N1E-115 cells and A7r5 cells were 3.0 T 0.6 and 2.5 T 0.41, respectively. 4.2. Saponin-permeabilized N1E-115 cells exhibit high InsP3 sensitivity of Ca2+ release To assess the possibility of a diffusible cytosolic factor contributing to this difference in InsP3 sensitivity between A7r5 cells and N1E-115 cells, both cell types were permeabilized by saponin (to release potential cytosolic modulators), then the InsP3 dependence of Ca2+ release was examined, using radiochemical techniques. Specifically, saponin-permeabilized cells accumulated 45Ca for 1 min at 22 -C, then various concentrations of InsP3 (in the presence or absence of heparin) were added, followed 5 s later by vacuum filtration and washing of the filter with EGTA. The levels of heparin-inhibitable InsP3-induced Ca2+ release for the permeabilized cells are shown in Fig. 1C, with both permeabilized A7r5 cells and permeabilized N1E-115 cells exhibiting a high InsP3 sensitivity. Half-maximal Ca2+ release for saponin-permeabilized A7r5 cells and N1E-115 cells was observed at 0.08 T 0.02 AM InsP3 and 0.19 T 0.05 AM InsP3, respectively (Fig. 1D). This InsP3 sensitivity of permeabilized A7r5 cells (using 45Ca flux) approximates that observed in intact A7r5 cells (using Ca2+ imaging/caged InsP3 techniques), suggesting that the permeabilization did not exert adverse effects on InsP3-induced Ca2+ release. The increase in InsP3 sensitivity of Ca2+ release in N1E-115 cells after permeabilization, on the other hand, raises the possibility of a diffusible cytosolic inhibitor of Ca2+ release in intact N1E-115 cells. 4.3. Cytosol from N1E-115 cells contains inhibitors of InsP3-induced Ca2+ release Gel filtration of cytosol from N1E-115 cells yielded two prominent inhibitors of InsP3-induced Ca2+ release from saponin-permeabilized N1E-115 cells (Fig. 2A), labeled ‘‘a’’ and ‘‘b’’, along with two lesser peaks labeled ‘‘c’’ and ‘‘aa’’. An identical inhibition profile was obtained using saponinpermeabilized A7r5 cells which contain predominantly peripheral type 1 InsP3 receptors (unfilled triangles in Fig. 2A), indicating that the inhibitors are not specific for neuronal type 1 InsP3 receptors which are prominent in

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Fig. 1. Neuroblastoma cells require high concentrations of InsP3 to induce Ca2+ release, whereas permeabilization of N1E-115 cells increases InsP3 sensitivity. Panels (A) and (B) represent the rises in intracellular Ca2+ in smooth muscle A7r5 cells and neuroblastoma N1E-115 cells, respectively, following uv photolysis of caged InsP3 within the cells. Rises in intracellular Ca2+ were monitored using the Ca2+ indicator calcium green – 10 kDa dextran. Arrows show times of uncaging, with calculated AM uncaged InsP3. Scale bars represent 20 s. Panel (C) compares the InsP3 dependence of Ca2+ release from intact N1E-115 cells (filled circles) and intact A7r5 cells (filled triangles) from the uncaging experiments shown in panels (A) and (B) with that obtained from saponinpermeabilized N1E-115 cells (unfilled circles) and saponin-permeabilized A7r5 cells (unfilled triangles). Ca2+ release from permeabilized cells was measured using 45Ca flux. The concentrations of InsP3 required for half-maximal Ca2+ release (i.e., K0.5) are shown in panel (D).

neuroblastoma cells [11]. The apparent molecular weights of inhibitory fractions ‘‘b’’ and ‘‘c’’ were 8 kDa and 800 Da, respectively, whereas inhibitory peaks ‘‘a’’ and ‘‘aa’’ eluted in the void volume (i.e., >30 kDa). The protein profile during the elution of N1E115 cytosol is shown in Fig. 2B, with peak protein concentration co-eluting with the lesser inhibitory peak ‘‘aa’’. The predominant inhibitory fraction ‘‘a’’ was subsequently applied to a superose 12 column and eluted at an apparent molecular weight of 400 kDa (Fig. 2C). Inhibitory peaks ‘‘a’’ and ‘‘b’’ decreased InsP3-induced Ca2+ release in a concentration-dependent manner, as shown in Fig. 3A. Curve fitting analyses indicated Hill coefficients of 2 – 3, suggesting that binding of 2 –3 molecules of inhibitor is needed for inhibition of the channel. Inhibitory peaks ‘‘a’’ and ‘‘b’’ were also shown to decrease the InsP3 sensitivity of Ca2+ release in permeabilized cells (Fig. 3B). Similarly, the 400 kDa inhibitory peak from the superose 12 column (which represented a more purified form of inhibitory peak ‘‘a’’) also decreased the InsP3 sensitivity of Ca2+ release from permeabilized cells (data not shown). To assess the possibility that the above inhibition of InsP3-induced Ca2+ release was due to InsP3 sequestration, inhibitory fractions ‘‘a’’ and ‘‘b’’ and the 400 kDa eluate were directly assayed for InsP3 binding. Using media comparable to that used for the inhibition of InsP3-induced Ca2+ release, none of these three inhibitory fractions bound InsP3 (as shown in Fig. 3C). As an internal control for the

binding assay, cerebellar microsomes were shown to bind 2000 cpm 3H-InsP3 (equivalent to 12 pmol InsP3/mg protein) under identical assay conditions, with this binding completely blocked by the competitive inhibitor heparin. Given the data in Figs. 3A and B, inhibition constants were calculated and used to predict the InsP3 dependence of Ca2+ release in the presence of each inhibitor (‘‘a’’ and ‘‘b’’) at a concentration thought to exist in intact N1E-115 cells (i.e., undiluted N1E-115 cytosol). These calculations predict that intact N1E-115 cells contain cytosolic concentrations of inhibitors ‘‘a’’ and Fb’’ to require approximately 10 AM InsP3 for half-maximal Ca2+ release. Thus, levels of inhibitors ‘‘a’’ and ‘‘b’’ in N1E-115 cytosol appear to be more than sufficient to account for the low InsP3 sensitivity of Ca2+ release observed in intact N1E-115 cells. In fact, the high level of inhibitory activity observed in these two peaks (‘‘a’’ and ‘‘b’’) raises the possibility that some inhibitor may be bound/sequestered in the intact N1E-115 cell. 4.4. Further purification of the ‘‘high molecular weight’’ cytosolic inhibitor(s) of InsP3-induced Ca2+ release from N1E-115 cells We have shown in Fig. 2A that N1E-115 cytosol contains a high molecular weight inhibitor of Ca2+ release (labeled ‘‘a’’), which eluted at 400 kDa upon superose 12 gel filtration (Fig. 2C). This predominant 400 kDa inhibitor was

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applied to the superose 12 column. As shown in Fig. 4D, the inhibitory peak eluted near the end of the column volume (below the molecular weight resolution). SDS-polyacrylamide gel electrophoresis of the inhibitory fractions showed the doublet of ¨73 kDa, along with some 10 kDa band (Fig. 4E). Densitometry of the gels showed that the level of the 73 kDa doublet was higher in inhibitory fractions (Fig. 4F) compared to non-inhibitory fractions near peak A3, whereas the level of the 10 kDa band was equivalent in inhibitory

Fig. 2. Elution of N1E-115 cytosol from a superdex 30 gel filtration column yields high and low molecular weight inhibitors of InsP3-induced Ca2+ release (A). Column eluates were assayed for inhibition of InsP3-induced Ca2+ release from permeabilized N1E-115 cells (filled circles) and permeabilized A7r5 cells (unfilled triangles), yielding two prominent inhibitory peaks ‘‘a’’ and ‘‘b’’ (A). Panel (B) shows the elution profile of protein from the superdex 30 column. Panel (C) shows the elution of inhibitory peak ‘‘a’’ from a superose 12 gel filtration column.

also observed when N1E-115 cytosol was applied directly to the superose 12 column (peak labeled ‘‘a1’’ in Fig. 4A). The ¨400 kDa inhibitor ‘‘a1’’ was then applied to a MonoQ column (equilibrated with 20 mM NaCl), yielding two inhibitory fractions eluting at 150 mM NaCl and 200 mM NaCl (well before much of the protein eluted), along with a lesser inhibitory peak at 300 mM NaCl (Fig. 4B). SDSpolyacrylamide gel electrophoresis of the two predominant inhibitory fractions (eluting at 150 mM and 200 mM NaCl) did not show high molecular weight bands but instead showed Coomassie blue bands at 10 kDa, 29 kDa, and a doublet of ¨73 kDa (Fig. 4C). The latter electrophoretic profile raised the possibility that the superose 12 inhibitory peak ‘‘a1’’ may represent an aggregate or that the inhibitory factors are associated with high molecular proteins. The prominent inhibitor from the MonoQ column (i.e., eluting in fractions 4 and 5, at 150 –200 mM NaCl) was therefore re-

Fig. 3. The inhibitory peaks ‘‘a’’ (filled circles) and ‘‘b’’ (unfilled triangles) inhibited InsP3-induced Ca2+ release from A7r5 cells in a concentrationdependent manner (A) and decreased the InsP3 sensitivity of Ca2+ release (B). The unfilled circles in panel (B) represent the control InsP3-induced Ca2+ release from permeabilized A7r5 cells (i.e., in the presence of elution buffer), whereas the solid line and dotted line on the right of panel (B) represent the predicted InsP3 sensitivity of Ca2+ release in the presence of intracellular levels of inhibitors ‘‘a’’ and ‘‘b’’ in intact N1E-115 cells. Inhibitors ‘‘a’’ and ‘‘b’’ and the 400 kDa fraction did not bind 3H-InsP3 (C), whereas control cerebellar microsomes bound 3H-InsP3 under identical assay conditions in a heparin-sensitive manner.

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Fig. 4. Purification of the high molecular weight cytosolic inhibitor. Panel (A) shows the elution of N1E-115 cytosol from a superose 12 column, yielding a 400 kDa inhibitor of InsP3-induced Ca2+ release (labeled ‘‘a1’’). Anion exchange chromatography of ‘‘a1’’ yielded a prominent inhibitor of InsP3-induced Ca2+ release (‘‘a2’’ in panel B), which showed Coomassie blue staining bands at 10 kDa, 29 kDa, and a doublet of ¨73 kDa (C). The inhibitor ‘‘a2’’ was re-applied to the superose 12 gel filtration column (D) but eluted near the end of the column volume (labeled ‘‘a3’’), yielding silver stained bands at ¨73 kDa (doublet) and 10 kDa (E). The abundance of the ¨73 kDa doublet and the 10 kDa band in inhibitory fractions in ‘‘a3’’ and adjacent non-inhibitory fractions was assessed by densitometry of the silver stained gel (F). The dotted lines in panels (A) and (B) represent protein elution. InsP3-induced Ca2+ release was monitored using permeabilized A7r5 cells.

and non-inhibitory fractions. A plot of inhibition of InsP3induced Ca2+ release versus level of the 73 kDa doublet for fractions within/adjacent to peak A3 yielded a positive correlation (r = 0.7; data not shown), indicating that inhibitory activity was related to the abundance of the 73 kDa doublet. These data implicate the 73 kDa doublet in the inhibition of InsP3-induced Ca2+ release, though the latter correlation coefficient of 0.7 indicates that the level of the 73 kDa doublet does not completely explain the inhibitory activity of this fraction. As indicated in Discussion, additional factors also appear to be important. 4.5. Tandem MS/MS analysis of the ¨73 kDa doublet and 10 kDa band from N1E-115 cells The ¨73 kDa doublet (Fig. 4E) was subjected to in-gel trypsin digestion followed by tandem MS/MS. As shown in Table 1, these MS analyses identified the ¨73 kDa doublet as stress-induced phosphoprotein 1 (accession number BC003794; Table 1), based on 8 peptide fragments. The

molecular mass (MH+) and sequence of each peptide fragment, along with the locations of the peptides within this phosphoprotein, are indicated in Table 1. The 10 kDa band from the gel shown in Fig. 4D (fractions 4 and 5) was also analyzed by tandem MS/MS and attributed to eukaryotic translation initiation factor 2A (eIF2A; accession number AK009934). This identification was based on 1 peptide fragment (Table 1), with the sequence and location within the initiation factor indicated in red. 4.6. Immunoprecipitation with mSTI1 antibody decreases inhibitory activity As indicated in Fig. 4A, gel filtration of N1E-115 cytosol on a superose 12 column yielded a prominent inhibitory peak at 400 kDa. To assess the contribution of mSTI1 to this inhibition, aliquots of this peak were subjected to immunoprecipitation with a monoclonal antibody to mSTI1, then the supernatant was assayed for inhibition of InsP3-induced

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Table 1 Identification of the protein bands in the inhibitory fractions by mass spectrometry

Ca2+ release from permeabilized A7r5 cells. To control for dilution of the 400 kDa inhibitor and/or non-specific effects of the immunoprecipitation protocol, identical aliquots of the inhibitory fraction were subjected to immunoprecipitation using either buffer or a control antibody (anti-Hsp72/ 73) instead of anti-mSTI1, then the supernatant was assayed for inhibition of Ca2+ release. As shown in Fig. 5, permeabilized A7r5 cells released 60% of the ionomycinsensitive Ca2+ pool upon addition of 0.7 AM InsP3. The 400 kDa fraction from N1E-115 cytosol, however, completely inhibited this InsP3-induced Ca2+ release. This complete inhibition was apparent whether the 400 kDa fraction was subjected to the control immunoprecipitation in the absence of a primary antibody or presence of a control antibody to Hsp72/73 (Fig. 5). If, however, the 400 kDa fraction was

subjected to immunoprecipitation in the presence of antibody to mSTI1, the supernatant only partially inhibited InsP3-induced Ca2+ release. That is, approximately twothirds of the InsP3-induced Ca2+ release was restored following immunoprecipitation of mSTI1, indicating that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release by N1E-115 cytosol. 4.7. Comparison of N1E-115 cells and A7r5 cells To ascertain if these inhibitory fractions are specific for cells exhibiting low InsP3 sensitivity, cytosol from A7r5 cells was subjected to gel filtration (identical to that used for cytosol from N1E-115 cells) and then assayed for inhibitory activity. As shown in Fig. 6, fractionation of cytosol from

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Fig. 5. Immunoprecipitation of mSTI1 from N1E115 cytosol decreases the inhibition of InsP3-induced Ca2+ release. The 400 kDa inhibitory peak (comparable to peak ‘‘a1’’ in Fig. 4A) was subjected to immunoprecipitation using antibodies to mSTI1 or Hsp72/73 or in the absence of primary antibody (as specified), then the supernatant was assayed for inhibition of InsP3-induced Ca2+ release from permeabilized A7r5 cells. ‘‘400 kDa/ GFB’’ and ‘‘400 kDa/PBS’’ represent the use of gel filtration buffer or phosphate-buffered saline instead of primary antibody in the immunoprecipitation protocol. Control represents the extent of InsP3-induced Ca2+ release in the absence of the 400 kDa inhibitory peak.

neuroblastoma cells. Results from this study are consistent with this hypothesis and raise the possibility that the cytosolic protein stress-induced phosphoprotein 1 (BC003794) may contribute to the low InsP3 sensitivity of Ca2+ release observed in differentiated N1E-115 cells. The issue of InsP3 sensitivity is important as a low InsP3 sensitivity in neurons could facilitate the integration of synaptic inputs (and/or promote co-incidence detection). This is particularly important for cells such as the cerebellar Purkinje neuron, which is innervated by up to 250,000 parallel fibers. Activation of parallel fibers can induce longterm depression of the Purkinje neuron, a process that requires activation of metabotropic glutamate receptors on the Purkinje neuron and subsequent Ca2+ release from InsP3-sensitive Ca2+ stores in the Purkinje cell spine [8,20,24]. Thus, a low InsP3 sensitivity provides the Purkinje neuron with the ability to integrate synaptic inputs (i.e., increasing the level of InsP3 within the spine) before initiating a change in synaptic plasticity. Distal dendrites of cortical pyramidal neurons have also been shown to exhibit a low InsP3 sensitivity [35], which may contribute to the integration of synaptic inputs from the various cortical layers [35,36].

A7r5 cells on a superose 12 column did show the presence of a small amount of inhibitory activity in fractions eluting at ¨6.5 ml and ¨9.5 ml (filled circles in A), though this was considerably less than that observed from an equivalent of cytosol from N1E115 cells (unfilled circles in panel A). The predicted molecular weight of inhibitory peak ‘‘b’’ (eluting at ¨9.5 ml) is ¨400 kDa, whereas the molecular weight of inhibitory peak ‘‘a’’ is beyond the resolution of the column (>700 kDa). The elution of A7r5 cytosolic protein from the superose 12 column is shown in Fig. 6B. 4.8. Western blot analysis of mSTI1 in N1E-115 cells and A7r5 cells As our data implicate mSTI1 in the inhibition of InsP3induced Ca release, the levels of mSTI1 were measured in N1E-115 cells and A7r5 cells, using Western blot techniques. Both A7r5 cells and N1E-115 cells contained mSTI1 (Fig. 7A), though the level of mSTI1 was 50% higher in N1E-115 cells (Fig. 7B). The calculated molecular weight of the chemiluminescent band was 66 kDa, which approximates the published molecular weight of mSTI1 of 62.5 kDa (accession # BC003794).

5. Discussion The basic question addressed in the present study is whether cytosolic components could explain the low InsP3 sensitivity of Ca2+ release seen in some neurons and

Fig. 6. Gel filtration of cytosol from A7r5 cells (filled circles in panel A) yielded low levels of inhibitors of InsP3-induced Ca2+ release compared to that observed in N1E-115 cells (unfilled circles in panel A). Equivalent amounts of cytosol from A7r5 cells and N1E-115 cells were applied to the superpose 12 column, then eluates were assayed for inhibition of InsP3induced Ca2+ release using permeabilized A7r5 cells. The unfilled triangles with a dashed line in panel (A) represent the elution of molecular weight standards. Inhibitory peak ‘‘b’’ eluted at ¨400 kDa, whereas inhibitory peak ‘‘a’’ eluted in the void volume. The elution of A7r5 cytosolic protein is shown in panel (B).

J. Watras et al. / Brain Research 1055 (2005) 60 – 72

Fig. 7. Western blot analysis of mSTI1 in homogenates of A7r5 cells (lanes 1 – 4) and differentiated N1E-115 cells (lanes 5 – 8). The chemiluminescent signals are shown in panel (A). The amount of protein applied to the lanes was: 77 Ag (lanes 1, 5); 38.5 Ag (lanes 2, 6); 19.25 Ag (lanes 3, 7); and 9.62 Ag (lanes 4, 8). The intensity of the chemiluminescent signals for lanes 2 – 4 (filled circles) and lanes 6 – 8 (unfilled circles) is plotted in panel (B). The chemiluminescent signal from the 77 Ag lanes was excluded from the regression in panel (B) due to the trail of chemiluminescence above the bands. The calculated molecular weight of the chemiluminescent bands in panel A is 66 kDa.

The low InsP3 sensitivity of distal dendrites in cortical pyramidal neurons has been hypothesized to result from a high density of InsP3 receptors [35], although reports on neuroblastoma cells [11] and hippocampal CA1 neurons [29] suggest otherwise. Neurites of neuroblastoma cells, for example, also exhibited a lower InsP3 sensitivity of Ca2+ release than the soma, but this was not associated with a high density of InsP3 receptors. Instead, the neurites had a 40 –60% lower density of ER and InsP3 receptors than the soma [11]. In fact, it has been speculated that a high density of InsP3 receptors may facilitate Ca2+ release [29] since Ca2+ release from one channel could act as a co-agonist for InsP3-induced Ca2+ release from an adjacent InsP3-gated Ca2+ channel. The low InsP3 sensitivity of Ca2+ release from neurons and neuroblastoma cells also does not appear to result from an inherent property of the neuronal InsP3 receptors. Specifically, the neuronal type 1 InsP3 receptor predominates in cerebellar Purkinje cells [34] and differentiated N1E1115 neuroblastoma cells [11], though purified

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reconstituted neuronal type InsP3-gated Ca2+ channel exhibits high sensitivity to InsP3 [6,18]. Half-maximal rate of InsP3-induced Ca2+ release from immunopurified reconstituted neuronal type 1 InsP3-gated Ca2+ channels, for example, occurred at ¨100 nM InsP3 [18], comparable to the InsP3 sensitivity of Ca2+ release from A7r5 cells or permeabilized N1E-115 cells shown in the present study. These results raise the possibility that the low InsP3 sensitivity of Ca2+ release in cerebellar Purkinje cell neurons and N1E-115 neuroblastoma cells may be due to cytosolic modulators of the InsP3-gated Ca2+ channel. InsP3 sensitivity of Ca2+ release can be modulated by cAMP-dependent phosphorylation [4,39], though half-maximal Ca2+ release of unphosphorylated channels was observed at 0.2 AM InsP3, with cAMP-dependent phosphorylation resulting in Ca2+ release at <0.1 AM InsP3. Thus, the low InsP3 sensitivity of intact N1E-115 cells [11] or cerebellar Purkinje neurons [24] does not appear to be due to the phosphorylation status of the channel. The type of InsP3 receptor isoform also affects InsP3 sensitivity [40], with type 2 InsP3 receptors having the highest InsP3 sensitivity and type 3 the lowest. The neuronal type 1 InsP3 receptor (which predominates in cerebellum [37] and N1E-115 cells [11]) exhibits half-maximal Ca2+ release at 0.1– 0.2 AM InsP3 [7,19] when isolated from the cell. In intact N1E-115 cells and cerebellar Purkinje neurons, half-maximal Ca2+ release occurred at 1 –3 AM and 20 AM (respectively) [11,13,24], which is 10- to 100fold higher than the concentration of InsP3 needed to activate purified InsP3 receptors. Moreover, the high InsP3 sensitivity of N1E-115 cells upon saponin permeabilization (as shown in Fig. 1) argues against differential expression of receptor isoforms as a contributing factor to the low InsP3 sensitivity in intact N1E-115 cells and cerebellar Purkinje neurons. There have been a few reports of cytosolic inhibitors of InsP3-induced Ca2+ release, the first of which raised the possibility that calmodulin may participate in the low InsP3 sensitivity of Ca2+ release in neurons [31]. It was hypothesized that neurons contain sufficiently elevated levels of calmodulin (compared to that in peripheral cells) to competitively inhibit InsP3-induced Ca2+ release. We cannot rule out possible contributions of calmodulin to the low InsP3 sensitivity of Ca2+ release in neurons and/or neuroblastoma cells, though our results point to the involvement of a different protein (mSTI1) in a prominent inhibitory fraction from N1E-115 cytosol. We previously reported that a low molecular weight (¨1600 Da) competitive inhibitor of InsP3 binding could be isolated from cerebellar microsomes, cerebellum, and neuroblastoma cells but was below detection in aortic smooth muscle [42]. We speculated that this low molecular weight inhibitor was a sulfated glycan. The present study shows evidence of a low molecular weight inhibitor (¨800 Da) of InsP3-induced Ca2+ release in N1E-115 cytosol,

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though the two prominent inhibitors were of considerably higher molecular weight (Fig. 2A). Hirata et al. [17] isolated a high molecular weight glycoprotein (>390 kDa) from rat brain cytosol that inhibited InsP3 binding. Although superose 12 gel filtration did suggest the presence of a cytosolic inhibitor with an apparent molecular weight of 400 kDa, further purification yielded a 73 kDa doublet identified as stress-induced phosphoprotein 1 (accession #BC003794; mSTI1) and a 10 kDa band attributed to eukaryotic translation initiation factor 2A (AK009934; eIF2A), suggesting that the 400 kDa inhibitory peak was an aggregate. The level of inhibition of InsP3-induced Ca2+ release by fractions within the 400 kDa peak correlated with the level of the 73 kDa doublet (r = 0.7), whereas the level of 10 kDa band was comparable in inhibitory and non-inhibitory fractions. These results raise the possibility that mSTI1 within the 400 kDa peak contributes to the inhibition of InsP3-induced Ca2+ release. Stress-induced phosphoprotein 1 (mSTI1) is a cochaperone that binds heat shock proteins 70 and 90 [27] and may play a role in refolding denatured proteins [22]. There are ten potential tetratricopeptide repeat (TPR) domains within mSTI1, important for protein – protein interactions, and six potential phosphorylation sites [3,30]. The mSTI1 shuttles between the nucleus and cytoplasm, depending on the phosphorylation status of mSTI1 [28]. Cellular mSTI1 may also bind to prion protein (PrPc) in the cell membrane and inhibit neuronal apoptosis [43]. Thus, there are multiple sites of mSTI1 binding, one of which appears to be anti-apoptotic. mSTI1 inhibition of InsP3induced Ca2+ release, as suggested by results from the present study, may provide another means of inhibiting apoptosis, given reports that InsP3-mediated Ca2+ release may promote apoptosis [2,5,21]. It is interesting to note that the region of the gel in Fig. 4E used for the tandem MS/MS identification of mSTI1 included both bands in the 60– 73 kDa region, though the fragmentation pattern indicated the presence of only one protein (mSTI1). As mSTI1 contains multiple phosphorylation sites, the doublet at 60– 73 kDa may represent unphosphorylated and phosphorylated forms of mSTI1. The results from the immunodepletion studies (shown in Fig. 5) are consistent with a role of mSTI1 in the inhibition of InsP3-induced Ca2+ release. It is likely, however, that the 400 kDa fraction used in these immunodepletion studies contains an additional inhibitor(s) of InsP3-induced Ca2+ release, as suggested by the subsequent purification steps, where the 400 kDa fraction yielded two inhibitory peaks following anion exchange chromatography (cf. Fig. 4B). The inability of the immunoprecipitation procedure to completely eliminate the inhibitory activity of the 400 kDa fraction is also consistent with the presence of an additional inhibitor(s). The observation that two-thirds of the inhibitory activity could be removed by the immunoprecipitation of mSTI1, however, does implicate mSTI1 as a

predominant inhibitor in this cytosolic fraction. Further study is required to ascertain if mSTI1 binds directly to the InsP3R or if an accessory protein(s) is required (e.g., using yeast 2-hybrid assays and/or GST fusion protein pull-down assays). Western blots of mSTI1 indicated that N1E-115 cells contain a 50% higher level of mSTI1 than do A7r5 cells, which is considerably less than the 10-fold difference in InsP3 sensitivity between these two cell types. Although this discrepancy suggests that mSTI1 is not a predominant inhibitor of InsP3-induced Ca2+ release in N1E-115 cells, an alternative explanation may involve the localization of mSTI1 in the two cell types. Specifically, calculations in Fig. 3 indicate that N1E-115 cells contain 3 – 10 times more cytosolic inhibitor than required to account for the low InsP3 sensitivity of Ca2+ release in intact N1E-115 cells, suggesting that much of the inhibitor(s) may be bound and/or sequestered. Moreover, the distribution of mSTI1 can change within a cell, with phosphorylation of mSTI1 at serine 189 promoting nuclear localization, whereas phosphorylation at threonine 198 promoted cytoplasmic localization [28]. Further studies are required to ascertain possible differences in the distribution/phosphorylation of mSTI1 in A7r5 cells and N1E-115 cells, along with the influence of mSTI1 phosphorylation on InsP3 sensitivity. The phospholipase C related catalytically inactive protein p130 [38] has been shown to competitively inhibit InsP3induced Ca2+ release, apparently by binding InsP3. Although the cytosolic inhibitors isolated from N1E-115 cells did not show evidence of a 130 kDa protein, attention was directed at the possibility that the cytosolic inhibitors may bind InsP3 (and thus reduce InsP3 concentration rather than the InsP3 sensitivity of the Ca2+ channel). As shown in Fig. 3, there was no evidence of InsP3 binding by the cytosolic inhibitors of Ca2+ release isolated from N1E-115 cells. Similarly, there was no evidence of Ca2+ binding by any of the cytosolic inhibitors (data not shown). Carbonic anhydrase related protein (CARP) has been shown to associate with the cerebellar InsP3 receptor and can inhibit InsP3 binding [19]. As cerebellar Purkinje cells contain CARP [23], the low InsP3 sensitivity of Ca2+ release in Purkinje neurons could be attributable at least in part to CARP. The apparent molecular weight of CARP is 35 kDa, and although a protein of ¨29 kDa was apparent upon partial purification of one of the cytosolic inhibitors, this protein peak was not apparent in a more purified preparation of the inhibitor (Fig. 4E). In summary, the present study provides evidence that the low InsP3 sensitivity of Ca2+ release from N1E-115 cells is attributable to the presence of diffusible cytosolic inhibitors. The calculated intracellular levels of each of two prominent cytosolic inhibitory peaks were more than sufficient to account for the low InsP3 sensitivity of Ca2+ release in N1E115 cells, suggesting that some of the inhibitors may be sequestered/bound. Further purification procedures, tandem

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MS/MS analyses, and immunoprecipitation studies indicate that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release in neuroblastoma cells.

Acknowledgments Supported through grants from the National Institutes of Health National Center for Research Resources grant RR13186 (LL) and the National Institutes of Neurological Diseases and Stroke grant RO1 NS040158 (JW). Dr. David Han is gratefully acknowledged for the tandem MS/MS analyses. Comments/suggestions by Drs. Barbara E. Ehrlich and Ion Moraru during the course of this study are gratefully acknowledged.

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