INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The inositol (1,4,5)-trisphosphate receptor (InsP₃R) is an intracellular calcium (Ca²⁺) release channel that plays an important role in Ca²⁺ signaling in cells (Berridge, 1993). Three mammalian isoforms of the InsP₃R share 60-70% amino acid homology and differ in tissue distribution (Furuichi et al., 1994). The type 1 receptor (InsP₃R1) is a predominant neuronal isoform that plays an important role in brain function (Matsumoto et al., 1996) and contributes to synaptic plasticity (Fujii et al., 2000; Itoh et al., 2001). The InsP₃R plays a central role in signal transduction and is subjected to multiple levels of regulation (Berridge, 1993; Bezprozvanny and Ehrlich, 1995; Ferris and Snyder, 1992b; Furuichi et al., 1994; Taylor, 1998). Binding of InsP₃ triggers the InsP₃R channel opening. The activity of InsP₃R1 is biphasically modulated by cytosolic Ca²⁺ (Bezprozvanny et al., 1991; Finch et al., 1991; Iino, 1990; Kaznacheyeva et al., 1998; Ramos-Franco et al., 1998b) and allosterically potentiated by adenine nucleotides (Bezprozvanny and Ehrlich, 1993; Ferris et al., 1990; Iino, 1991). The InsP₃R1 is also phosphorylated by protein kinase A (PKA), protein kinase C (PKC), and Ca²⁺/calmodulin (CaM)-kinase (Ferris et al., 1991b; Supattapone et al., 1988; Yamamoto et al., 1989) with resulting changes in the InsP₃R1 function (Cameron et al., 1995; Nakade et al., 1994; Supattapone et al., 1988).

The functional InsP₃R channel is a tetrameric complex (Maeda et al., 1991; Mignery et al., 1989). Each InsP₃R subunit consists of three distinct domains (Mignery and Sudhof, 1990; Miyawaki et al., 1991): the carboxy-terminal Ca²⁺ channel domain; the amino-terminal ligand binding domain; and the middle coupling domain. A number of putative modulatory sites (phosphorylation sites, ATP binding sites, calmodulin binding site, Ca²⁺-binding sites) are located in the coupling domain of the InsP₃R1 (Furuichi et al., 1994). The SII site of alternative splicing is also located in this region (Furuichi et al., 1994). The predominant neuronal isoform of InsP₃R1 is SII(+) and nonneuronal isoform is SII() (Danoff et al., 1991; Nakagawa et al., 1991a, 1991b). The excision of the SII insert changes the PKA phosphorylation pattern of the InsP₃R1 (Danoff et al., 1991; Ferris et al., 1991a) and creates additional ATP (Ferris and Snyder, 1992b) and CaM (Islam et al., 1996; Lin et al., 2000) binding sites in the InsP₃R1 sequence.

The autosomal recessive opisthotonos (opt) is a spontaneous mouse mutation resulting in epileptic-like behaviors, similar to the phenotype of InsP₃R1 knockout mice (Matsumoto et al., 1996). The seizures in opt homozygotes begin at 14 days postnatal and become progressively more severe, leading to death at 3-4 weeks of age. Recent genetic analysis of the opt mutant identified a >10-kilobase (kb) deletion within the InsP₃R1 gene (Street et al., 1997). As a result of this deletion, a fragment of 107 amino acids, containing several putative regulatory sites, is removed from the InsP₃R1 coupling region in the opt mice (Street et al., 1997). Alterations in the InsP₃R1 properties caused by the opt mutation have not been previously described.

Here we analyzed single-channel behavior of the InsP₃R1-SII(+), InsP₃R1-SII(), and InsP₃R1-opt channels in identical experimental conditions. The recombinant InsP₃R1 for these studies were expressed in insect Spodoptera frugiperda (Sf9) cells using a baculovirus expression system. Microsomes isolated from the InsP₃R1-expressing Sf9 cells were fused to planar lipid bilayers, and activity of the InsP₃R1 was analyzed at the single channel level. Obtained results provided novel information about molecular determinants of the InsP₃R1 function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Generation of recombinant baculoviruses

The full-length neuronal rat InsP₃R1 (SI/SII+) (Mignery et al., 1990) expression construct in pcDNA3 vector was previously described (Kaznacheyeva et al., 1998). The coding sequence of the InsP₃R1-SII(+) was excised from pcDNA3 vector with XhoI and XbaI and subcloned into SalI and XbaI sites of pFastBac1 expression vector (Invitrogen Corp, Carlsbad, CA). Generated pFastBac1-InsP₃R1-SII(+) plasmid was transformed into DH10Bac (Invitrogen) Escherichia coli strain, and baculoviruses expressing InsP₃R1 were generated using Bac-to-Bac baculovirus expression system according to manufacturer's (Invitrogen) protocol. Generated RT1 (InsP₃R1-SII(+)) baculoviruses were amplified three times to yield P3 stock with the titer 10⁸-10⁹ pfu/ml. InsP₃R1-SII() (deletion of amino acids Q1692-R1731) and InsP₃R1-opt (deletion of amino acids G1732-Q1839) mutations were introduced by inverse polymerase chain reaction and verified by sequencing. The 2.5-kb fragments of InsP₃R1 sequence containing the SII() spliced region or the opt mutation were subcloned into pFastBac1-InsP₃R1 and the recombinant baculoviruses SII() (InsP₃R1-SII()) and opt (InsP₃R1-opt) were generated and amplified using Bac-to-Bac system (Invitrogen).

Expression of the InsP₃R1 in Sf9 cells

Sf9 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in suspension culture in supplemented Grace's insect media (Invitrogen) with 10% fetal bovine serum at 27°C. For the InsP₃R1 expression, 150 ml of Sf9 cell culture was infected by InsP₃R1-encoding baculovirus at 5-10 multiplicity of infection (MOI). 66 h post-infection, Sf9 cells were collected by centrifugation at 4°C for 5 min at 800 rpm (GH 3.8 rotor, Beckman Instruments, Fullerton, CA). The cellular pellet was resuspended in 25 ml of homogenization buffer (sucrose 0.25 M, Hepes 5 mM, pH 7.4) supplemented with protease inhibitors cocktail (1mM ethylenediaminetetraacetic acid, aprotinin 2 µg/ml, leupeptin 10 µg/ml, benzamidine 1 mM, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 2.2 mM, pepstatin 10 µg/ml, phenylmethyl sulfonyl fluoride 0.1 mg/ml). Cells were disrupted by sonication (Branson Ultrasonics, Danbury, CT) and manually homogenized on ice with a glass-Teflon (DuPont, Wilmington, DE) homogenizer. The microsomes were isolated from the Sf9 cell homogenate by gradient centrifugation as previously described for human embryonic kidnet (HEK)-293 cells (Kaznacheyeva et al., 1998). The final microsomal preparation was resuspended in 0.5 ml of the storage buffer (10% sucrose, 10 mM 3-(N-Morpholino)propanesulfonic acid pH 7.0) to typically yield 6 mg/ml of protein (Bradford assay, Bio-Rad, Hercules, CA), aliquoted, quickly frozen in liquid nitrogen, and stored at 80°C. Expression of the InsP₃R1 was confirmed by Western blotting using the anti-InsP₃R1 rabbit polyclonal antibody that was previously described (Kaznacheyeva et al., 1998).

Single-channel recordings and analysis of the InsP₃R1 activity

Recombinant InsP₃R1 expressed in Sf9 cells were incorporated into the bilayer by microsomal vesicle fusion as described previously for native cerebellar InsP₃R and for the InsP₃R1 expressed in HEK-293 cells (Bezprozvanny and Ehrlich, 1993, 1994; Bezprozvanny et al., 1991; Kaznacheyeva et al., 1998). Single-channel currents were recorded using 50 mM Ba²⁺ dissolved in Hepes (pH 7.35) in the trans (intraluminal) side as a charge carrier (Bezprozvanny and Ehrlich, 1994). Transmembrane potential during current recordings was fixed to 0 mV in Ca²⁺- and ATP-dependence experiments, and varied between +10 mV and 30 mV in current-voltage relationship experiments. The cis (cytosolic) chamber contained 110 mM Tris dissolved in Hepes (pH 7.35). To obtain Ca²⁺-dependence of the InsP₃R1, we followed the protocol from Bezprozvanny et al. (1991). Free Ca²⁺ concentration in the cis chamber was controlled in the range of 10 nM (pCa 8) to 10 µM (pCa 5) by a mixture of 1 mM EGTA, 1 mM N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid, and variable concentrations of CaCl₂. The resulting free Ca²⁺ concentration was calculated by using a program described in Fabiato (1988). ATP-dependence of InsP₃R1 was measured by consecutive addition of Na₂ATP to the cis chamber from 100 mM stock. All additions (InsP₃, ATP, CaCl₂) were to the cis chamber from the concentrated stocks with at least 30 s of stirring solutions in both chambers. InsP₃R1 single-channel currents were amplified (Warner OC-725, Warner Instruments Corp, Hamden, CT), filtered at 1 kHz by low-pass 8-pole Bessel filter, digitized at 5 kHz (Digidata 1200, Axon Instruments, Union City, CA) and stored on a computer hard drive and recordable optical discs.

For off-line computer analysis (pClamp 6, Axon Instruments) single-channel data were filtered digitally at 500 Hz; for presentation of the current traces, data were filtered at 200 Hz. Evidence for the presence of 2-3 functional channels in the bilayer was obtained in majority of the experiments. The number of active channels in the bilayer was estimated as a maximal number of simultaneously open channels during the course of an experiment (Horn, 1991). The open probability of closed level, and 1st and 2nd open levels was determined by using half-threshold crossing criteria (t  2 ms) from the records lasting at least 2.5 min. The single-channel open probability (Po) for one channel was calculated using the binomial distribution for the levels 0, 1, and 2, and assuming that the channels were identical and independent (Colquhoun and Hawkes, 1995). In analysis of Ca²⁺- and ATP-dependence experiments, potential errors in absolute Po values were minimized by normalizing the Po to the maximum Po observed in the same experiment.

Ca²⁺ imaging in DT40 cells

DT40 chicken B lymphoma cells were cultured in RPMI1640 supplemented with 10% fetal calf serum, 1% chicken serum, penicillin (100 U/ml), streptomycin (100 U/ml), and 2 mM glutamine. Mutant DT40 cells with all three of their InsP₃R genes disrupted (Sugawara et al., 1997) were transfected with the linearized rat pcDNA3-InsP₃R1-SII(+), pcDNA3-InsP₃R1-SII(), and pcDNA3-InsP₃R1-opt plasmids by electroporation (330 V, 250 µF). Several stably expressing clones were isolated in the presence of 2 mg/ml G418 (Geneticin, Invitrogen). Ca²⁺ imaging of the InsP₃R1-SII(+), InsP₃R1-SII(), and InsP₃R1-opt transfected cells was performed as described previously (Miyakawa et al., 1999, 2001). Briefly, cells on poly-L-lysine and collagen-coated coverslips were loaded with 1 µM Fura-2AM. The fluorescence images were captured at room temperature (22-24°C) with an Olympus IX70 inverted microscope, equipped with a cooled charge-coupled device camera (Photometrics, Tucson, AZ) and a polychromatic illumination system (T.I.L.L. Photonics, Germany) at a rate of one pair of frames with excitation at 345 and 380 nm every 10, 1, or 0.25 s. Intracellular Ca²⁺ concentrations of the Fura-2-loaded cells were calculated using the equation reported previously (Grynkiewicz et al., 1985).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Functional expression of the InsP₃R1-SII(±) splice variants and the InsP₃R1-opt mutant

To study the properties of recombinant InsP₃R1, we generated baculovirus encoding rat InsP₃R1-SII(+) (RT1), InsP₃R1-SII() (SII()), and InsP₃R1-opt (opt) as described in Methods. The SII splicing region of InsP₃R1 is 40 amino acids long and can be further subdivided into A, B, and C regions (Nakagawa et al., 1991a, 1991b). The sequence of InsP₃R1-SII(+) (Mignery et al., 1990) (Fig. 1 A) corresponds to the SIIAC isoform, a major cerebellar isoform of the InsP₃R1 (Nakagawa et al., 1991a, 1991b). The sequence of InsP₃R1-SII() (Fig. 1 A) corresponds to SIIABC() isoform (deletion of Q1692-R1731) expressed in peripheral tissues (Danoff et al., 1991; Nakagawa et al., 1991a, 1991b). Genomic deletion in the opt mutant results in removal of two exons immediately after the SII region of alternative splicing (Street et al., 1997). As a result of alternative splicing in the SIIABC region, four possible InsP₃R1 mRNAs are expressed in brains of opt homozygotes (Street et al., 1997). In this paper we re-created the opt mutation (deletion of G1732-Q1839) on the basis of InsP₃R1-SIIAC isoform (Fig. 1 A).

View larger version (26K): [in this window] [in a new window]   FIGURE 1   Structure and expression of the InsP3R1-SII(+) (RT1), InsP3R1-SII() (SII()), and InsP3R1-opt (opt) isoforms. (A) Diagram of rat InsP3R1-SII(+), InsP3R1-SII(), and InsP3R1-opt constructs used in this study. The domain structure of the InsP3R1, ATP-binding sites, CaM-binding site, transmembrane domains, and the Ca2+ channel pore region are adapted from Furuichi et al. (1994). The preferential sites of PKA phosphorylation (S1589 for SII() and S1755 for SII(+)) are from Danoff et al. (1991) and Ferris et al. (1991a). PKA phosphorylation pattern of the InsP3R1-opt mutant is not known. The E2100 residue was recently identified as part of the InsP3R1 activating Ca2+ sensor (Miyakawa et al., 2001). (B) Western blot of microsomal proteins. Rat cerebellum microsomes (cer) and microsomes isolated from Sf9 cells infected with RT1, SII(), and opt baculoviruses were analyzed by Western blotting with anti-InsP3R1 polyclonal antibody. For each microsomal preparation, 20 µg of total protein was loaded on the gel.

Microsomes isolated from the RT1, SII() and opt-infected Sf9 cells, but not from noninfected cells, contained large quantities of the InsP₃R1 detectable by Western blotting (Fig. 1 B). Small amounts of endogenous InsP₃R1 were detected in microsomes from noninfected Sf9 cells when the blots were overexposed (data not shown). The apparent molecular size of recombinant InsP₃R1-SII(+) was identical to the InsP₃R1 present in rat cerebellar microsomes (Fig. 1 B). The predicted molecular weights are 311,401 Da for the InsP₃R1-SII(+), 306,939 Da for the InsP₃R1-SII(), and 300,299 Da for the InsP₃R1-opt. Relatively small size differences among the InsP₃R1-SII(+) and the InsP₃R1-SII() (1.4%) or the InsP₃R1-opt (3.6%) isoforms could not be reliably resolved on the 8% acrylamide gel used in our experiments. The shorter molecular size products detected by anti-InsP₃R1 antibodies in all four samples (Fig. 1 B) correspond to partial degradation products of the InsP₃R1 resulting from the limited proteolysis during microsomal extraction or isolation procedure. From the relative abundance of degradation products on the gel, it seems that the InsP₃R1-opt mutant is more sensitive to proteolysis than the wild type SII(+) or SII() isoforms (Fig. 1 B). It is possible that the increased sensitivity of InsP₃R1-opt mutant to proteolysis contributed to 10-fold reduction in the level of the InsP₃R1 protein in the brain of the opt mutant mice (Street et al., 1997).

When microsomes isolated from the RT1-infected Sf9 cells were fused with planar lipid bilayers, InsP₃-gated channels were frequently (in 30 of 40 experiments) observed (Fig. 2). In contrast, the InsP₃-gated channels were never (n = 10) observed in experiments with microsomes from noninfected cells. Therefore, we concluded that channels observed in our planar lipid bilayer experiments with microsomes from the RT1-infected Sf9 cells correspond to the activity of recombinant rat InsP₃R1. The InsP₃R1 plasmid used to generate RT1 baculovirus corresponds to a major cerbellar isoform SIIAC (Fig. 1 A). As expected, the gating and conductance properties of channels observed in experiments from RT1-infected Sf9 cells were identical to the native channels observed in experiments with rat cerebellar microsomes (Fig. 2). To determine the functional properties of the InsP₃R1-SII() splice variant and the InsP₃R1-opt mutant channels, we fused microsomes from the Sf9 cells infected with SII() and opt baculoviruses with planar lipid bilayers. In both cases the InsP₃-gated channels were recorded (Fig. 2). The gating behavior of the InsP₃R1-SII() splice variant and the InsP₃R1-opt mutant channels was similar to the behavior of InsP₃R1-SII(+) and native cerebellar InsP₃R, but the unitary current was different (Fig. 2). Indeed, in identical recording conditions the InsP₃R-SII() channels supported larger current, and the InsP₃R1-opt channels supported smaller current than the InsP₃R-SII(+) channels (Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIGURE 2   Single-channel records of native rat cerebellar InsP3R, recombinant InsP3R1-SII(+), InsP3R1-opt, and InsP3R1-SII() isoforms in planar lipid bilayers. Ca2+ and ATP act as co-agonists of the InsP3R1, but are not able to activate the channels without InsP3 present (first traces). Addition of 2 µM InsP3 to the cis (cytoplasmic) side activates the InsP3R1 (third traces). Current traces at the expanded time scale are shown on the bottom.

Gating and conductance properties

Systematic analysis of the InsP₃R1 functional properties (Fig. 3, Table 1) revealed that the mean open times of the channels formed by native cerebellar InsP₃R, the InsP₃R1-SII(+) isoform, and the InsP₃R1-opt mutant are all close to 5 ms and are not significantly different from one another. Compared with other isoforms, the mean open time of the InsP₃R1-SII() channels is elevated by 46% to 7.3 ms (Fig. 3, Table 1). The size of the unitary current for native cerebellar InsP₃R and the InsP₃R1-SII(+) splice variant was 2.0 pA. The size of the unitary current was increased to 2.3 pA for the InsP₃R1-SII(+) splice variant and reduced to 1.6 pA for the InsP₃R1-opt mutant (Fig. 3). Statistical analysis (Table 1) revealed that the unitary current supported by the InsP₃R1-SII() splice variant is significantly larger and the current supported by the InsP₃R1-opt mutant is significantly smaller than the current supported by the native cerebellar InsP₃R or recombinant InsP₃R1-SII(+) isoform.

View larger version (13K): [in this window] [in a new window]   FIGURE 3   Analysis of the single channel records obtained with native rat cerebellar InsP3R (cer), recombinant InsP3R1-SII(+) (RT1), InsP3R1-opt (opt), and InsP3R1-SII() (SII()) isoforms. Open dwell time (left) distributions and unitary current histogram (right) are shown. Open time distributions were fit with a single exponential function (curve) that yielded 0 of 5.5 ms for native rat cerebellar InsP3R, 0 of 4.7 ms for InsP3R1-SII(+), 0 of 5.2 ms for InsP3R1-opt, and 0 of 7.3 ms for InsP3R1-SII(). Unitary currents were fitted with a Gaussian function that was centered at 2.0 pA for rat cerebellar InsP3R, 1.9 pA for InsP3R1-SII(+), 1.6 pA for InsP3R1-opt, and 2.3 pA for InsP3R1-SII(). The figure was generated with the data from the same experiments as shown in Fig. 2. Similar analysis of three independent experiments with each InsP3R1 isoform was performed to generate the data presented in Table 1.

View this table: [in this window] [in a new window]   TABLE 1   Comparison of basic single-channel properties of native rat cerebellar and recombinant InsP3R1-SII(+), InsP3R1-SII(), and InsP3R1-opt forms of the InsP3R1

To further characterize the conductance properties of different InsP₃R1 isoforms, we determined the unitary current supported by these channels at various transmembrane potentials between + 10 mV and 30 mV (Fig. 4). The slope of the resulting current-voltage relationship provided us with the value of single-channel conductance equal to 80 pS for the native cerebellar InsP₃R, 81 pS for the InsP₃R1-SII(+), 94 pS for the InsP₃R1-SII(), and 64 pS for the InsP₃R1-opt (Fig. 4, Table 1). Thus, we concluded that the single-channel conductance of the InsP₃R1-SII() splice variant is significantly higher and the conductance of the InsP₃R1-opt mutant is significantly lower than the single-channel conductance of native cerebellar InsP₃R or the recombinant InsP₃R1-SII(+) isoform.

View larger version (13K): [in this window] [in a new window]   FIGURE 4   Current-voltage relationship of native rat cerebellar InsP3R (cer), recombinant InsP3R1-SII(+) (RT1), InsP3R1-opt (opt), and InsP3R1-SII() (SII()) isoforms. Activities of these InsP3R1 isoforms were recorded in planar lipid bilayers in the range of transmembrane potentials from 30 mV to +10 mV. Single-channel current amplitude at each voltage was determined from a Gaussian fit. The data sets were fit by a linear regression (r = 0.99) with a slope of 80 pS for cerebellar InsP3R (), 81 pS for InsP3R-SII(+) (), 64 pS for InsP3R1-opt (), and 94 pS for InsP3R1-SII() (). All points represented mean (±SE; n  3).

Modulation by cytosolic calcium

Bell-shaped dependence of the InsP₃R1 on cytosolic Ca²⁺ (Bezprozvanny et al., 1991; Finch et al., 1991; Iino, 1990) is one of the fundamental InsP₃R1 properties responsible for complex spatiotemporal aspects of Ca²⁺ signaling (Berridge, 1993). In the next series of experiments we evaluated the modulation of recombinant rat InsP₃R1-SII(+), the InsP₃R-SII(), and the InsP₃R1-opt mutant by cytosolic Ca²⁺. In agreement with the behavior of native cerebellar InsP₃R ((Bezprozvanny et al., 1991) and Fig. 5, ) and recombinant rat InsP₃R1 expressed in HEK-293 and COS cells (Kaznacheyeva et al., 1998; Ramos-Franco et al., 1998a), recombinant InsP₃R1-SII(+) expressed in Sf9 cells displays bell-shaped Ca²⁺ dependence with the maximal open probability at 300 nM Ca²⁺ (Fig. 5, ). The channels formed by the InsP₃R1-SII() splice isoform (Fig. 5, ) and by the InsP₃R1-opt mutant (Fig. 5, ) also display bell-shaped Ca²⁺ dependence with the peak at 300 nM Ca²⁺. The bell-shaped Ca²⁺ dependence of recombinant InsP₃R1-SII(+) was wider than the bell-shaped Ca²⁺ dependence of cerebellar InsP₃R (Fig. 5) or the bell-shaped Ca²⁺ dependence of InsP₃R1 expressed in HEK-293 cells (Kaznacheyeva et al., 1998). In contrast, the bell-shaped Ca²⁺-dependence of InsP₃R1-SII() and InsP₃R1-opt forms was even narrower than the Ca²⁺-dependence of native cerebellar InsP₃R1 (Fig. 5). To obtain quantitative description of these differences, Ca²⁺-dependence of different InsP₃R1 isoforms was fitted by the bell-shaped equation from (Bezprozvanny et al., 1991) (Fig. 5, smooth curves). The parameters of the optimal Ca²⁺-dependence fit for all four InsP₃R1 isoforms are presented in Table 2. The reasons for the differences in the shape of Ca²⁺-dependence of different InsP₃R1 forms in our experiments are not entirely clear (see Discussion).

View larger version (20K): [in this window] [in a new window]   FIGURE 5   Bell-shaped Ca2+ dependence of native rat cerebellar InsP3R (cer), recombinant InsP3R1-SII(+) (RT1), InsP3R1-opt (opt), and InsP3R1-SII() (SII()) isoforms. The open-channel probability of the InsP3R1 (Po) was determined in the presence of 2 µM InsP3, 0.5 mM Na2ATP, and the cis (cytosolic) Ca2+ concentrations in the range between 10 nM and 5 µM Ca2+. P0 in each experiment was normalized to maximum P0 observed in the same experiment, and then data from several independent experiments were averaged together at each Ca2+ concentration. The normalized and averaged data at each Ca2+ concentration are shown as mean ± SE (n  3) for rat cerebellar InsP3R (), InsP3R1-SII(+) (), InsP3R1-opt (), and InsP3R1-SII() (). These data were fitted by the bell-shaped equation P(Ca2+) = Pm kn[Ca2+]n/((kn + [Ca2+]n)(Kn + [Ca2+]n)) from Bezprozvanny et al. (1991), where n is a Hill coefficient, k is the apparent affinity of Ca2+ activating site, and K is the apparent affinity of Ca2+ inhibitory site. The parameters of the best fit (smooth curves) are presented in Table 2.

View this table: [in this window] [in a new window]   TABLE 2   Parameters of bell-shaped fit to the Ca2+-dependence data obtained with the rat cerebellar InsP3R1 and recombinant InsP3R1-SII(+), InsP3R1-SII(), and InsP3R1-opt isoforms

To further evaluate the Ca²⁺ regulation of the InsP₃R1-SII() splice isoform and the InsP₃R1-opt mutant form, we compared Ca²⁺ signals induced by B cell receptor stimulation in DT40 cells transfected with the InsP₃R1-SII(+), InsP₃R1-SII(), and InsP₃R1-opt constructs. The temporal pattern of Ca²⁺ signals in DT40 cells expressing the InsP₃R1-SII(+) and InsP₃R1-opt was indistinguishable (Fig. 6), confirming the conclusion from bilayer experiments that opt mutation has only minimal effect on the InsP₃R1 modulation by Ca²⁺. Similar conclusion has been reached in the previous Ca²⁺ imaging studies of Purkinje neurons in opt mouse cerebellar slices (Street et al., 1997). A similar response was recorded in DT40 cells expressing InsP₃R1-SII() (data not shown). A sensitivity of Ca²⁺ imaging experiments in DT40 cells is not sufficient to detect changes in Ca²⁺ release properties resulting from the described above differences in single-channel conductance between the different InsP₃R1 isoforms.

View larger version (15K): [in this window] [in a new window]   FIGURE 6   Ca2+ signals in DT40 cells expressing InsP3R1-SII(+) (RT1) and InsP3R1-opt (opt) in response to BCR stimulation. The data are shown for 4 min (left) and 60 min (right). The anti-BCR antibody (1 µg/ml) was applied as indicated by the horizontal bars below the traces.

Modulation by ATP

The activity of the InsP₃R1 is allosterically potentiated by millimolar concentrations of adenine nucleotides (Bezprozvanny and Ehrlich 1993; Ferris et al., 1990; Iino, 1991). Two ATP-binding sites (ATPA and ATPB) are present in the InsP₃R1-SII(+) sequence (Ferris and Snyder, 1992a; Furuichi et al., 1994; Maes et al., 2001, 1999) (Fig. 1 A). The ATPA site is deleted in the InsP₃R1-opt mutant (Fig. 1 A). An additional putative ATP-binding site (ATPC) is created in the InsP₃R1-SII() splice variant by excision of SII insert (Ferris and Snyder, 1992b) (Fig. 1 A). What effect do these changes of the InsP₃R1 sequence have on its modulation by ATP? To answer this question, in the next series of experiments we compared the ATP-dependence of recombinant rat InsP₃R1-SII(+), the InsP₃R1-SII(), and the InsP₃R1-opt mutant. In agreement with the behavior of native cerebellar InsP₃R1 ((Bezprozvanny and Ehrlich, 1993); Fig. 7, ), the activity of recombinant InsP₃R1-SII(+) expressed in Sf9 cells was allosterically potentiated by ATP with the apparent affinity k_ATP of 0.24 mM ATP (Fig. 7, ). The sensitivity of the InsP₃R-opt mutant was reduced 20-fold, with the apparent affinity k_ATP of 5.3 mM (Fig. 7, ). The effect of opt mutation on ATP-dependence of InsP₃R1 is consistent with the location of the ATPA site in the InsP₃R1 sequence (Fig. 1 A).

View larger version (18K): [in this window] [in a new window]   FIGURE 7   Allosteric potentiation of native rat cerebellar InsP3R (cer), and recombinant InsP3R1-SII(+) (RT1), InsP3R1-opt (opt), and InsP3R1-SII() (SII()) isoforms by ATP. The single-channel open probability was measured as a function of Na2ATP concentration on the cytoplasmic side of the membrane. The ATP concentration ranged from 0 to 3.0 mM. At least 100 s of continuous recording at each concentration for ATP were analyzed to obtain the open probability. The open probability was normalized to the maximal open probability observed in the same experiment. The normalized data from several experiments with each InsP3R1 isoform were averaged together and plotted as mean ± SE (n  3) for native cerebellar InsP3R (), InsP3R1-SII(+) (), InsP3R1-opt (), and InsP3R1-SII() (). The data were fit by the equation P([ATP]) = P + Pm[ATP]/(kATP + [ATP]) from Bezprozvanny and Ehrlich (1993), where P is the open probability in the absence of ATP, Pm is the maximal increase in Po induced by ATP, and kATP is the apparent affinity for ATP. The parameters of the best fit (smooth curves) are in Table 1.

The alternative splicing of SII fragment had a dual effect on ATP-sensitivity of the InsP₃R1. First, when compared with the InsP₃R1-SII(+) isoform, the apparent affinity for ATP k_ATP was reduced sixfold in the InsP₃R1-SII() isoform to 1.33 mM (Fig. 7, ). Second, the InsP₃R1-SII() channels were twofold more active than InsP₃R1-SII(+) channels in the absence of ATP. On average, at 0 ATP P_o of the InsP₃R1-SII(+) channels was 6 ± 2% (n = 3), and P_o of the InsP₃R1-SII() channels was 13 ± 2% (n = 3). Similar conclusion was apparent from fitting the ATP-dependence data (Fig. 7, ); the ratio of P_o in the absence of ATP (P) to maximal P_o predicted by the ATP-dependence equation (P + P_m) is 0.19 for the InsP₃R1-SII(+) isoform and 0.27 for the InsP₃R1-SII() isoform (Table 1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this paper we compared the main functional properties of native rat cerebellar InsP₃R1, recombinant rat InsP₃R1-SII(+) and InsP₃R1-SII() splice variants, and recombinant InsP₃R1-opt deletion mutant. The properties of the channels were analyzed using planar lipid bilayer technique in identical experimental conditions. Recombinant InsP₃R1 for these studies were expressed in Sf9 cells using baculovirus-mediated infection. From obtained results we concluded that: 1) the properties of recombinant InsP₃R1-SII(+) channels expressed in Sf9 cells follow most of the properties of the native cerebellar InsP₃R1; 2) the InsP₃R1-SII() splice variant has higher conductance (94 pS) and the InsP₃R1-opt mutant has lower conductance (64 pS) than the InsP₃R1-SII(+) isoform (81 pS); 3) the mean open channel time is ~5 ms for the InsP₃R1-SII(+) and InsP₃R1-opt isoforms and 7.3 ms for the InsP₃R1-SII() isoform; 4) all three InsP₃R1 isoforms display bell-shaped Ca²⁺-dependence on cytosolic Ca²⁺ with the peak at 200-300 nM Ca²⁺; 5) the bell-shaped Ca²⁺-dependence is wider for the InsP₃R1-SII(+) isoform when compared with the InsP₃R1-SII() and InsP₃R1-opt isoforms, indicating possible differences in cooperative interaction between InsP₃R1 subunits; 6) all three InsP₃R1 isoforms support similar pattern of Ca²⁺ signals when expressed in DT40 cells; 7) when compared with the InsP₃R1-SII(+) isoform, the sensitivity to modulation by ATP is 20-fold lower in the InsP₃R1-opt mutant and sixfold lower in the InsP₃R1-SII() splice variant; 8) when compared with InsP₃R1-SII(+), the activity of InsP₃R1-SII() in the absence of ATP is elevated twofold. The main results of this paper are summarized in Table 1 and briefly discussed below.

Our finding that an alternative splicing (SII) or deletion (opt) in the coupling domain has an effect on single-channel conductance of the InsP₃R1 was unexpected. According to the conventional model of the InsP₃R domain structure, the structural determinants of channel pore are localized to the carboxy-terminal Ca²⁺ channel domain (Mignery and Sudhof, 1990; Miyawaki et al., 1991). From our results it seems that the middle portion of the coupling domain is intimately involved in the function of the InsP₃R pore. The amino-terminal and carboxy-terminal regions of InsP₃R have been shown to associate directly in biochemical experiments (Boehning and Joseph, 2000a; Joseph et al., 1995), and it is possible that the middle portion of the coupling domain is localized in the proximity of the channel pore in the three-dimensional structure of the InsP₃R. Interestingly, the effect of SII splicing on channel conductance seems more pronounced when divalent cations are used as current carrier. In our experiments with 50 mM Ba²⁺ as a current carrier, the single channel conductance of the InsP₃R1-SII() isoform was elevated by 16% (94 pS for SII() versus 81 pS for SII(+) (Fig. 4), whereas in experiments of Boehning et al. (2001) with 140 mM K⁺, the difference between single-channel conductance values of InsP₃R1-SII splice variants was only 5% (390 pS for SII() vs 370 pS for SII(+)). Changes in InsP₃R1 conductance induced by SII splicing event are not likely to be associated with the change in the InsP₃R1 PKA-phosphorylation pattern (Danoff et al., 1991; Ferris et al., 1991a) (Fig. 1 A). In an independent series of experiments we established that only ~20% of the InsP₃R1 expressed in Sf9 cells are in the PKA-phosphorylated state and that the single channel conductance of the InsP₃R1-SII(+) channels is not influenced by PKA phosphorylation (Tang at al, submitted for publication).

We concluded that the peak of bell-shaped Ca²⁺-dependence located at pCa 6.6-6.7 for the InsP₃R1-SII(+), InsP₃R1-SII(), and InsP₃R1-opt (Fig. 5, Tables 1 and 2). In agreement with this finding, the smooth muscle cells expressing the InsP₃R-SII() isoform display Ca²⁺ dependence of InsP₃-induced Ca²⁺ release with the maximum at pCa 6.5 (Iino, 1990), similar to the cerebellar InsP₃R1-SII(+) isoform with the maximim at pCa 6.7 (Bezprozvanny et al., 1991). Using a Ca²⁺ flux assay with transfected COS cells, Boehning and Joseph (2000b) reported that the InsP₃R1-SII() isoform is significantly more sensitive to modulation by Ca²⁺ than InsP₃R1-SII(+), with the peak at 10-20 nM Ca²⁺ (pCa 8). However, the same group reported similar Ca²⁺ dependence of InsP₃R1-SII() and InsP₃R1-SII(+) isoforms in patch-clamp studies of InsP₃R expressed in a COS cell nuclear envelope (Boehning et al., 2001). Thus, most likely the discrepancy between our results and the data of Boehning and Joseph (2000) is caused by the difference in assays used to analyze the recombinant InsP₃R1-SII() function.

Although the peak of Ca²⁺-dependence was observed at pCa 6.6-6.7 for all InsP₃R1 isoforms tested in our study, the shape of Ca²⁺-dependence bell was wider for the InsP₃R1-SII(+) than for the InsP₃R1-SII() and InsP₃R1-opt isoforms (Fig. 5). Also, the Ca²⁺-dependence was wider for the InsP₃R1-SII(+) than for the native cerebellar InsP₃R1 (Fig. 5). To obtain quantitative description of these differences, Ca²⁺-dependence of different InsP₃R1 isoforms was fitted by the bell-shaped equation from (Bezprozvanny et al., 1991) (Fig. 5, smooth curves). The fitting procedure yielded Hill coefficient (n) of 1.56 for cerebellar InsP₃R, 1.22 for InsP₃R1-SII(+), 4.04 for InsP₃R1-SII(), and 2.37 for InsP₃R1-opt (Table 2). At the moment, we do not clearly understand the reasons for observed differences in the width of bell-shaped Ca²⁺ dependence, but it is possible that alternative splicing (SII-) or deletion (opt) in the InsP₃R1 coupling domain affects interactions between InsP₃R1 subunits. The reasons for the differences in the width of Ca²⁺-dependence of the InsP₃R1-SII(+) expressed in Sf9 cells and of the native cerebellar InsP₃R (Fig. 5) or InsP₃R1-SII(+) expressed in HEK-293 cells (Kaznacheyeva et al., 1998) are also not clear. The most likely possibility is related to the absence of an auxiliary protein, such as FKBP12, in Sf9 cells (Brillantes et al., 1994). Future studies will be required to clarify this issue. Parameters of our fitting procedure indicate that the apparent affinity of Ca²⁺-activating site is close to 0.4 µM Ca²⁺ for all 3 InsP₃R1 isoforms (Table 2), in agreement with location of the InsP₃R1 Ca²⁺ sensor region (Miyakawa et al., 2001) outside the area affected by SII splicing and opt mutation (Fig. 1 A). When compared with InsP₃R1-SII(+), the apparent affinity of Ca²⁺-inhibitory site is elevated twofold in InsP₃R1-SII() and InsP₃R1-opt isoforms (Table 2). Thus, a putative Ca²⁺-inhibitory site may be located close to the region affected by SII splicing and opt mutation.

The dramatic effect of opt mutation on ATP-dependence of InsP₃R1 is consistent with the location of the ATPA site in the InsP₃R1 sequence (Fig. 1 A). In our experiments the apparent affinity for potentiation by ATP is reduced 20-fold in the InsP₃R1-opt mutant (Fig. 7, Table 1). The remaining sensitivity to ATP modulation in the InsP₃R-opt mutant is likely to be conferred by the intact ATPB site (Fig. 1 A). Notably, the ATPA site is unique for the InsP₃R1 isoform, whereas the ATPB site is conserved among InsP₃R1, InsP₃R2, and InsP₃R3 isoforms (Furuichi et al., 1994). When InsP₃R1 and InsP₃R3 isoforms were compared in Ca²⁺ flux studies, at least 10-fold reduction in sensitivity to ATP modulation has been observed for the InsP₃R3 isoform (Maes et al., 2000; Missiaen et al., 1998; Miyakawa et al., 1999). Thus, our data with the InsP₃R1-opt mutant support the notion that the ATPA site is responsible for high-affinity ATP binding and the affinity of the ATPB site is at least 10-fold lower (Maes et al., 2001).

The effect of SII splicing on ATP sensitivity is more complex. The apparent affinity to ATP potentiation is reduced approximately sixfold in the nonneuronal InsP₃R1-SII() isoform (Fig. 7, Table 1). In contrast, a level of basal activity in the absence of ATP is elevated twofold for the InsP₃R1-SII() isoform (Fig. 7, Table 1). In principle, our data agree with the previous description of the adenine nucleotide effect on InsP₃-induced Ca²⁺ release in smooth muscle cells (Iino, 1991), but detailed quantitative comparison is difficult. The ATPA site is intact in InsP₃R1-SII() isoform (Fig. 1 A), and the observed changes is likely to be attributable to overall changes in the InsP₃R1 coupling domain conformation induced by the SII splicing event. It is also possible that an additional ATPC site created in InsP₃R1-SII() isoform by SII excision (Ferris and Snyder, 1992b) (Fig. 1 A) is inhibitory, leading to reduction in the apparent affinity of InsP₃R1 for ATP.

Our data provide some new information related to the opisthotonos mouse phenotype. In agreement with conclusions of Street et al. (1997), we established that the InsP₃R1 containing the opt mutation is functional. The major cause of the opisthotonos phenotype is likely to be an impairment of Ca²⁺ release from intracellular stores resulting from a 10-fold reduction in level of the InsP₃R1 protein in the brain of opisthotonos mouse (Street et al., 1997). Interestingly, the InsP₃R1-opt mutant expressed in Sf9 cells seems to be more prone to proteolysis than the wild-type InsP₃R1 isoforms (Fig. 1 B). It is possible that the increased sensitivity of InsP₃R1-opt mutant to proteolysis is linked to reduction in the level of the InsP₃R1 protein in the brain of the opt mutant mice (Street et al., 1997). In addition, we found that when compared with the wild-type InsP₃R1-SII(+), the single-channel conductance of the InsP₃R1-opt mutant is reduced by 20% (Fig. 4) and sensitivity to potentiation by ATP is reduced 20-fold (Fig. 7). These changes in InsP₃R1 properties did not have a significant effect on BCR response when InsP₃R1-opt mutant was expressed and tested in DT40 cells (Fig. 6) but, in the brain, alterations in InsP₃R1 properties may contribute to severity of the opisthotonos mouse phenotype.