Control of IP3-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin

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Control of IP₃-mediated Ca²⁺ puffs in Xenopus laevis oocytes by the Ca²⁺-binding protein parvalbumin

Linu M. John, Monica Mosquera-Caro , Patricia Camacho † and James D. Lechleiter

Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908, * Department of Cellular and Structural Biology and † Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA

MS 12176 Received 15 January 2001; accepted after revision 20 March 2001

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Elementary events of Ca²⁺ release (Ca²⁺ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP₃Rs) at low concentrations of IP₃. Ca²⁺ puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP₃ into the cell. However, cells appear to have sufficient concentrations of IP₃ (0.1-3.0 µM) to induce Ca²⁺ release under resting conditions.
Here, we investigated Ca²⁺ puff activity in non-stimulated Xenopus oocytes using confocal microscopy. The fluorescent Ca²⁺ dye indicators Calcium Green 1 and Oregon Green 488 BAPTA-2 were injected into oocytes to monitor basal Ca²⁺ activity.
In this preparation, injection or overexpression of parvalbumin, an EF-hand Ca²⁺-binding protein (CaBP), induced Ca²⁺ puffs in resting Xenopus oocytes. This activity was inhibited by heparin, an IP₃R channel blocker, and by mutation of the Ca²⁺-binding sites in parvalbumin.
Ca²⁺ puff activity was also evoked by injection of low concentrations of the Ca²⁺ chelator EGTA, but not by calbindin D_28k, another member of the EF-hand CaBP superfamily.
BAPTA and the Ca²⁺ indicator dye Oregon Green 488 BAPTA-1 evoked Ca²⁺ puff activity, while the dextran conjugate of Oregon Green 488 BAPTA-1 did not. These data indicate that a Ca²⁺ buffer must be mobile in order to increase Ca²⁺ puff activity.
Together, the data indicate that some IP₃Rs spontaneously release Ca²⁺ under resting concentrations of IP₃. These elementary Ca²⁺ events appear to be below the level of detection of current imaging techniques. We suggest that parvalbumin evokes Ca²⁺ puffs by coordinating the activity of elementary IP₃R channel openings.
We conclude that Ca²⁺ release can be evoked not only by hormone-induced increases in IP₃, but also by expression of mobile cytosolic CaBPs under resting concentrations of IP₃.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

IP₃ production is under the control of many hormones and neurotransmitters (Berridge, 1993; Putney & Bird, 1993; Clapham, 1995). Depending on the concentration of IP₃, different patterns of intracellular Ca²⁺ release can be induced by ligand binding to the IP₃R (Lechleiter & Clapham, 1992; Parker et al. 1996; Bootman et al. 1997b; Camacho & Lechleiter, 2000). At low concentrations of exogenously injected IP₃ (~50 nM), discrete Ca²⁺ release events, referred to as Ca²⁺ puffs, are frequently observed in Xenopus oocytes (Parker & Ivorra, 1990a; Parker et al. 1996) as well as other cell types (Kong et al. 1996; Reber & Schindelholz, 1996; Bootman et al. 1997b; Bobanovic et al. 1999; Koizumi et al. 1999). Single channel openings from individual IP₃Rs are referred to as Ca²⁺ blips (Parker et al. 1996). Paradoxically, spontaneous firing of the IP₃R channel has rarely been observed under non-stimulated conditions, even though resting concentrations of IP₃ have been reported to be as high as 100 nM in the oocyte and ~3 µM in other cell types (Bradford & Rubin, 1986; Horstman et al. 1988; Underwood et al. 1988; DeLisle et al. 1990; Bird et al. 1991; Stith et al. 1992; Luzzi et al. 1998). The absence of detectable Ca²⁺ puffs at resting levels of IP₃ could be due to desensitization of the IP₃Rs, the low amplitude of Ca²⁺ release events at rest or, alternatively, the compartmentalization of basal IP₃, which would prevent the ligand from activating IP₃Rs.

Parvalbumin is an EF-hand CaBP expressed at very high levels in muscle and neuronal cells (Berchtold et al. 1984; Hou et al. 1991b; Kosaka et al. 1993). In muscle tissues, parvalbumin has been shown to increase the rate of muscle relaxation by slowly binding Ca²⁺ (Gillis et al. 1982; Heizmann et al. 1982; Hou et al. 1991a; Andressen et al. 1995; Muntener et al. 1995; Jiang et al. 1996). Muscles of neuromuscular mutant mice with arrested development of the righting response exhibit reduced parvalbumin content and prolonged half-relaxation times (Stuhlfauth, 1984). Several neuromuscular pathologies are attributed to deficiencies in parvalbumin content (Pauls et al. 1996). Motor neurons enriched in parvalbumin are reportedly resistant to amyotrophic lateral sclerosis, a neurodegenerative motor neuron disease characterized by increased Ca²⁺ influx and intracellular Ca²⁺ concentrations (Appel et al. 1995; Elliott & Snider, 1995; Reiner et al. 1995; Ho et al. 1996). Parvalbumin has also been postulated to act as a cytosolic Ca²⁺ buffer that protects neurons from high cytotoxic Ca²⁺ (Miller & Baimbridge, 1983; Sloviter, 1989; Heizmann & Hunziker, 1991; Chard et al. 1993). Unlike pyramidal neurons, which do not express this CaBP, GABAergic hippocampal neurons survive ischaemic injury and cell death (Celio, 1988; Nitsch et al. 1989). The resistance to cell death is attributed to the Ca²⁺-buffering capacity of parvalbumin. Ca²⁺ buffering by parvalbumin is also proposed to facilitate efficient signalling by fast firing interneuronal cells (Kawaguchi et al. 1987). Although parvalbumin does not alter resting Ca²⁺ levels, it reduces the peak (Dreessen et al. 1996) and the rate of rise, and increases the fast component of the decay rate in Ca²⁺ transients produced by brief depolarizations in rat sensory neurons and in neuroblastoma cells (Chard et al. 1993).

In this report, we present evidence for a novel function of parvalbumin. We demonstrate that injection or overexpression of parvalbumin evokes Ca²⁺ puffs in the absence of exogenous injections of IP₃. We attribute the increase in Ca²⁺ activity to a parvalbumin-mediated action on IP₃Rs bound with resting concentrations of IP₃. Further, we show that this increase in Ca²⁺ puffs is dependent on parvalbumin's ability to bind Ca²⁺. Consequently, the expression and/or presence of parvalbumin must be considered as a factor that can enhance IP₃-mediated Ca²⁺ release under non-stimulated resting IP₃ levels

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Oocyte protocols

Xenopus oocytes were collected under anaesthesia from albino frogs that were humanely killed after the final collection. Stage V-VI oocytes were mechanically defolliculated and incubated at 18 °C in 43 % L-15 media (Gibco-BRL) until used for confocal imaging (Camacho & Lechleiter, 1995). Oocytes were injected using glass pipettes with a 10 µl Drummond micropipette. All injections were delivered as a 50 nl bolus. Final concentrations are reported for a 1:20 dilution assuming a 1 µl oocyte volume.

IP₃ assay

Resting IP₃ concentrations were determined using a [³H]IP₃ assay system, according to the instructions provided by the manufacturer (TRK1000, Amersham). Groups of 10 oocytes were homogenized in 10 % (v/v) perchloric acid and incubated on ice for 10 min. After centrifugation (2000 g, 15 min, 4 °C), the supernatant was added to a 1:1 mixture of Freon and tri-n-octylamine containing a final concentration of 2 mM EDTA. After an additional centrifugation, the neutralized supernatant was incubated with tritiated IP₃ and a suspension of bovine adrenal IP₃-binding proteins for 15 min. Following separation by centrifugation of bound from free IP₃, the resulting pellet was solubilized with NaOH and radioactivity was measured in the bound fraction. A standard curve was plotted from measurements of two sets of eight standards that were run with each of the experimental samples. Unlabelled IP₃ concentrations were calculated by interpolation from the standard curve. All measurements were made in triplicate.

Confocal imaging and analysis

Ca²⁺ activity was primarily imaged using a NORAN OZ laser scanning confocal microscope attached to a Nikon TE200 inverted microscope with a times 60, 1.2 NA water objective. The slit was set to 10 µm, the sampling time was 3200 ns (1.07 s per image), the screen size was 512 pixels times 480 pixels and the zoom was set at 1. For the EGTA imaging experiments, the sampling time was 6400 ns and the screen size was 256 pixels times 240 pixels. Some experiments were also performed on a Nikon PCM2000 confocal microscope using the same objective. Image analysis was performed with ANALYZE software (Mayo Clinic, Rochester, MN, USA) on a Silicon Graphics O2 workstation. Single-line scans were obtained at ~2.2 ms per line. Ca²⁺ increases are reported as $Delta$ F/F, which represents increments in fluorescence (F) with respect to resting values (F_rest) and is calculated as (F - F_rest)/F_rest. All images were acquired in extracellular medium containing 96 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 5 mM Hepes (pH 7.5) and 1 mM EGTA.

Expression vector construction

All cDNAs were subcloned between the 5' and 3' untranslated regions of Xenopus laevis $beta$ -globin as previously described (Camacho & Lechleiter, 1995). The polymerase chain reaction (PCR) was used to amplify the full open reading frame of the cDNAs encoding rat wild-type parvalbumin (P_wt) and a mutant parvalbumin deficient in Ca²⁺ binding (P_CDEF) (Epstein et al. 1986; Pauls et al. 1994). The forward primer had the sequence 5'-ACTGGGATCCATGTCGATGACAGACTTGCTC-3' and included a BamH I site at the NH₂-terminus, while the reverse primer, 5'-ACTGAAGCTTCGCCACTTAGCTTTCGGCC-3', incorporated a Hind III site at the 3' end of the P_wt- and P_CDEF-encoding cDNAs. Following amplification, the PCR product was gel isolated, digested with BamH I and Hind III, and subcloned into the vector pGEM-HE Not. The P_CDEF mutant has four amino acid substitutions replacing the first (D51A and E62V) and last (D90A and E101V) amino acids of the two metal-binding loops (Pauls et al. 1994). These highly conserved charged residues contain essential oxygen sites for metal binding. Automatic DNA sequencing (core facility, University of Texas Health Science Center at San Antonio) confirmed the correct sequence of the mutant and wild-type proteins.

In vitro transcription

Synthetic mRNAs were prepared as previously described (Camacho & Lechleiter, 1995). Briefly, plasmids were linearized with Not I and in vitro transcribed from the T7 promoter using the Megascript transcription kit according to the manufacturer's instructions (Ambion, Austin, TX, USA). Transcripts were also co-transcriptionally capped with m⁷G(5')ppp(5'') (Ambion). All synthetic mRNAs were resuspended at a concentration of 1.5-2.0 µg µl^-1 and stored in aliquots of 3 µl at -80 °C until used.

Protein detection

Overexpression of P_wt and P_CDEF in the oocytes was determined by radioactive labelling with [³⁵S]methionine. [³⁵S]methionine (10 µCi µl^-1, Tran ³⁵S label, New England Nuclear) was co-injected into oocytes with the appropriate mRNA. Control oocytes were injected with label alone. Groups of five oocytes were frozen at -80 °C after incubation for 8-9 h in extracellular medium containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM Hepes (pH 7.5) and 1.8 mM CaCl₂. Oocytes were later homogenized in lysis buffer (100 mM NaCl, 1 mM EDTA, 0.2 % BSA and 20 mM Tris, pH 7.6) supplemented with 1 mM phenylmethylsulfonyl fluoride and 5 µg ml^-1 each of pepstatin A and leupeptin (Schmalzing et al. 1997). The homogenate was centrifuged at 10 000 g for 30 s at 4 °C, and the resulting supernatant was centrifuged again at 100 000 g for 1 h at 4 °C. Cytosolic proteins contained in the final supernatant were precipitated with acetone. The final pellet from each extract was resuspended in 1 % SDS. Proteins were resolved by SDS-PAGE on a 16 % gel loaded with one or four oocyte equivalents for detection by autoradiography. Gels were fixed, dried and exposed to X-ray blue sensitive film (RPI) at -80 °C for 16 h.

Reagents

Purified Ca²⁺-binding proteins (rat parvalbumin and calbindin D_28K) were obtained from SWANT (Switzerland). Calcium indicator dyes were obtained from Molecular Probes (Eugene, OR, USA). All other reagents were purchased from Sigma.

Statistical analysis

Statistical significance was determined by using Student's one-tailed t test or a Chi-squared test as appropriate. Ca²⁺ puff frequency was calculated by counting and averaging Ca²⁺ puffs over a period of 60 s in individual oocytes.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Injection of parvalbumin protein evokes Ca²⁺ puffs

To examine the role of parvalbumin in Ca²⁺ signalling, oocytes were injected with purified protein (50 µM final concentration) together with the Ca²⁺ indicator dye Calcium Green 1 (12.5 µM final concentration). Confocal imaging was performed ~30-60 min later to allow for equilibration of both protein and dye. Unexpectedly, Ca²⁺ puff activity was immediately observed in 88 % of the oocytes (n = 33), in the absence of exogenously injected IP₃ (Fig. 1). The mean frequency of Ca²⁺ puffs in responding oocytes was 30.4 ± 9.0 events (100 s)^-1. When the injected parvalbumin was reduced 10-fold in concentration (5 µM final), a decrease in the number of responding oocytes (29 %, n = 7) and in the frequency of Ca²⁺ puff activity (9.5 ± 2.2 events (100 s)^-1) was observed. At 6-fold higher parvalbumin concentrations (300 µM final), the percentage of oocytes exhibiting Ca²⁺ puff activity decreased (46 %, n = 13). However, at this higher concentration the frequency of Ca²⁺ puffs increased to 160.8 ± 7.7 events (100 s)^-1. In contrast, control oocytes (10 %, n = 40) rarely exhibited Ca²⁺ puffs (1.0 ± 0.1 events (100 s)^-1) (Fig. 1C). These data demonstrate that the presence and frequency of Ca²⁺ puffs are sensitive to the concentration of parvalbumin.

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Figure 1. Parvalbumin induces Ca²⁺ puffs in the absence of IP₃ injections in Xenopus oocytes
A, spatio-temporal stacks of Ca²⁺ puff activity in oocytes injected with parvalbumin (~50 µM final). The left panel presents the spatial Ca²⁺ puff activity accumulated from 240 images collected at 1 s intervals. The average background was subtracted from each image and the resulting images were superimposed to give a single image using a maximum intensity projection algorithm. The right panel presents the temporal stack of the Ca²⁺ puff activity from the same data as in the left panel (rotated 90 deg). B, parvalbumin-induced spontaneous Ca²⁺ puffs are blocked by heparin (100 ng µl^-1 final), a competitive inhibitor of the IP₃R. C, dose dependence of parvalbumin-induced Ca²⁺ puff activity. Left panel shows the percentage of oocytes exhibiting Ca²⁺ puffs (see text for number of oocytes tested at each concentration of parvalbumin). Notice that a percentage of non-injected oocytes (10 %, n = 40) also exhibited puffs, which occurred at very low frequency (1 ± 0.01 events (100 s)^-1). Heparin (Hep) completely blocked Ca²⁺ puffs on all oocytes tested (n = 9).

Parvalbumin-evoked Ca²⁺ puffs are blocked by heparin

Ca²⁺ puffs are thought to represent the spatial-temporal summation of single channel IP₃R openings in isolated clusters of receptors (Parker et al. 1996; Bootman et al. 1997a; Horne & Meyer, 1997). IP₃ binding to the IP₃R is considered to be an essential requirement for channel opening (Meyer et al. 1988; Iino, 1990; Parker & Ivorra, 1990a; Watras et al. 1991). Consequently, when Ca²⁺ puffs were observed in the absence of a ligand-binding event at the plasma membrane, we hypothesized that the resting IP₃ concentration was responsible for the activation of a sub-population of IP₃Rs. Heparin, a well-characterized competitive inhibitor of IP₃ binding to the IP₃R (Worley et al. 1987; Kobayashi et al. 1989), was used to test whether the Ca²⁺ puffs evoked in the presence of parvalbumin were due to IP₃R activation. Oocytes were injected with heparin (100 ng µl^-1 final), Calcium Green 1 and parvalbumin, and were imaged (30-60 min later) to measure Ca²⁺ puff activity. Heparin completely blocked the ability of parvalbumin to evoke Ca²⁺ puffs (Fig. 1B and C). This indicates that the binding of IP₃ to the IP₃R is required for the Ca²⁺ puffs evoked by parvalbumin.

To rule out the possibility that the parvalbumin-evoked Ca²⁺ puff activity was due to an increase in the amount of ligand produced by the CaBP, we measured the levels of IP₃ in the presence and absence of parvalbumin (50 µM final, see Methods). When compared to control uninjected oocytes (75 ± 6 nM; n = 14 groups, 10 oocytes per group, pooled from 6 frogs), parvalbumin did not significantly change the concentration of IP₃ (68 ± 8 nM; n = 14 groups, 10 oocytes per group). Thus, parvalbumin must be evoking Ca²⁺ puffs without increasing the IP₃ concentration. The IP₃ concentrations we measured are comparable to those reported by other investigators in the Xenopus oocyte in the resting state (DeLisle et al. 1990; Stith et al. 1992; Luzzi et al. 1998). Furthermore, these values are higher than the known affinity of the receptor for IP₃, which is approximately 50 nM (Worley et al. 1987; Supattapone et al. 1988; Kaftan et al. 1997). Thus, it appears that there is sufficient ligand to activate the IP₃R at rest. We conclude that parvalbumin may evoke Ca²⁺ puffs by uncovering this activity without further increases in IP₃ concentration.

Expression of parvalbumin evokes Ca²⁺ puffs

Commercially available purified parvalbumin may contain a contaminating agent responsible for activating IP₃Rs. To rule out this potential artifact, we obtained the cDNA encoding rat parvalbumin (Epstein et al. 1986) and expressed it in Xenopus oocytes, which do not express parvalbumin endogenously (Kay et al. 1987). As observed with injections of purified protein, expression of parvalbumin caused spontaneous Ca²⁺ puffs in the absence of IP₃ injections. Indeed, the majority of oocytes exhibited Ca²⁺ puffs (78 %, n = 36) and the frequency of these events (30.4 ± 1.15 events (100 s^-1)) was comparable to that in oocytes injected with purified parvalbumin (50 µM final) (Fig. 2A-C). Ca²⁺ puffs were observed as early as 6 h following injection of mRNA, but rarely after 24 h. Taken together, these data indicate that parvalbumin itself is responsible for evoking Ca²⁺ puffs and that low levels of parvalbumin expression are necessary and sufficient to induce these Ca²⁺ puffs.

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Figure 2. Spontaneous Ca²⁺ puffs are induced by overexpression of parvalbumin
A, spatio-temporal stack of Ca²⁺ puff activity in oocytes injected with mRNA encoding wild-type parvalbumin. Images were acquired 12 h following mRNA injection. B, mutagenesis of the high affinity Ca²⁺-binding EF-hand motifs in parvalbumin (CDEF) inhibits the occurrence of spontaneous Ca²⁺ puffs. Ca²⁺ puff activity was collected 12 h after the oocyte was injected with the mRNA encoding the mutant parvalbumin. Background subtraction and analysis were performed as in Fig. 1. C, the percentage of oocytes displaying Ca²⁺ puffs is presented in the left histogram while the frequency of these events in those oocytes that had Ca²⁺ puffs is shown in the right histogram. Expression of wild-type parvalbumin (P_wt) produced Ca²⁺ puff activity that was indistinguishable from that of oocytes injected with 50 µM parvalbumin (Fig. 1C). Overexpression of the mutant parvalbumin (P_CDEF) reduced the percentage of oocytes exhibiting Ca²⁺ puffs (** P < 0.005) and lowered the frequency of puff activity in these oocytes (* P < 0.004). Results for P_CDEF were not significantly different from those for control oocytes (C). D, expression of P_wt and P_CDEF as determined by radioactive labelling with [³⁵S]methionine. P_CDEF migrates below P_wt evidently as a result of the mutation of four negatively charged amino acids in the Ca²⁺-binding domains to non-polar residues (see Methods).

Parvalbumin-evoked Ca²⁺ puffs are dependent on cytosolic Ca²⁺ buffering

In the absence of Mg²⁺, parvalbumin binds Ca²⁺ with a stoichiometry of two and with high affinity (K_D ~20 nM) (Pauls et al. 1994). If the occurrence of Ca²⁺ puffs is due to Ca²⁺ binding to parvalbumin, then mutagenesis of the Ca²⁺-binding sites in parvalbumin should inhibit Ca²⁺ puff activity. To test this hypothesis, we used a cDNA encoding a mutant of parvalbumin in which the two EF-hand Ca²⁺-binding domains were mutated to inhibit Ca²⁺ buffering (Pauls et al. 1994). We found that the percentage of oocytes responding with Ca²⁺ puffs (21 %, n = 19, P < 0.005) and the frequency of Ca²⁺ puff activity (4.1 ± 0.4 events (100 s)^-1, P < 0.004) were significantly reduced in oocytes that overexpressed the mutant parvalbumin as compared to the wild-type protein. In addition, the values obtained with the mutant parvalbumin were not statistically different from the Ca²⁺ puff activity exhibited by control oocytes, not injected with mRNA (Fig. 2B and C). To rule out the possibility that differences in protein levels accounted for the findings, we measured protein synthesis by metabolically labelling the oocytes with [³⁵S]methionine. Oocytes injected with mutant parvalbumin mRNA expressed protein at comparable levels to oocytes injected with wild-type parvalbumin mRNA (Fig. 2D). These data indicated that the ability of parvalbumin to evoke Ca²⁺ puffs was dependent on its EF-hand Ca²⁺-binding domain.

EGTA evokes Ca²⁺ puffs

Mg²⁺ ions bind to the Ca²⁺-binding sites located in parvalbumin, albeit with relatively low affinity (K_D ~100 µM) (Gillis et al. 1982; Hou et al. 1991b; Jiang et al. 1996). Because oocytes and many other cell types contain millimolar concentrations of Mg²⁺, these binding sites are occupied with Mg²⁺ under non-stimulated conditions (Chard et al. 1993). Consequently, parvalbumin could be evoking Ca²⁺ puffs by lowering the free Mg²⁺ concentration. Mg²⁺ has been reported to inhibit IP₃R activation and IP₃ binding non-competitively (Volpe et al. 1990). To determine whether this mechanism of action was responsible for the effects of parvalbumin, we injected oocytes with low concentrations of the Ca²⁺ chelator EGTA, which does not bind Mg²⁺ at physiological concentrations. We found that EGTA (25 µM final) significantly increased the proportion of oocytes exhibiting Ca²⁺ puff activity as compared to control oocytes (P < 0.005). Specifically, EGTA evoked activity in 11 % of the oocytes tested (n = 310), with a mean frequency of 9.3 ± 0.44 events (100 s)^-1 (n = 20, Fig. 3). When the EGTA concentration was lowered to 5 µM (final), Ca²⁺ puff activity occurred in only 6.6 % of tested oocytes (n = 106) with a mean frequency of 8.1 ± 0.59 events (100 s)^-1 (n = 11, Fig. 3B). At higher EGTA concentrations (50 µM final), 6.1 % of the oocytes (n = 114) exhibited Ca²⁺ puffs at a mean frequency of 4.3 ± 0.32 events (100 s)^-1 (n = 19). Ca²⁺ puff activity was completely abolished when the EGTA concentration was increased to 500 µM (final). Since EGTA does not lower the Mg²⁺ concentration, we conclude that low levels of Ca²⁺ buffering by EGTA, or parvalbumin, can evoke Ca²⁺ puffs under basal, non-hormone-stimulated concentrations of IP₃.

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Figure 3. Injection of low concentrations of EGTA in Xenopus oocytes induces Ca²⁺ puff activity
A, spatio-temporal stacks of Ca²⁺ puff activity in an oocyte injected with EGTA (25 µM final). The images were analysed and presented as in Fig. 1. The scale is the same as in Fig. 1B. B, histograms of Ca²⁺ puff activity show a dose dependency on the concentration of EGTA injected.

Parvalbumin reduces Ca²⁺ puff amplitude and duration

To further characterize the effects of parvalbumin, we imaged the time course of single Ca²⁺ puffs at a higher temporal resolution (Fig. 4). We found that parvalbumin-evoked Ca²⁺ puffs (n = 120) had significantly shorter decay rates (58 ± 2.3 ms, P < 0.001) and were of significantly smaller amplitude ( $Delta$ F/F = 0.34 ± 0.01, P < 0.0005) than Ca²⁺ puffs induced by exogenous IP₃ injections. In control oocytes injected with IP₃ (n = 54), Ca²⁺ puffs had a mean decay of 86.8 ± 3.7 ms and a $Delta$ F/F value of 0.41 ± 0.02 (Fig. 4C and D). Parvalbumin-induced Ca²⁺ puffs also exhibited a significantly faster rise time (33.0 ± 1.4 ms, P < 0.035) when compared to exogenous IP₃-induced Ca²⁺ puffs (37.1 ± 1.8 ms). The decreases in peak amplitude and decay time are consistent with increased cytosolic Ca²⁺ buffering by parvalbumin. A reduction in these parameters could also be explained by fewer IP₃-bound IP₃Rs contributing to Ca²⁺ puffs at resting levels of ligand. Faster rise times are consistent with a parvalbumin-mediated increase in Ca²⁺ release during the initial stages of Ca²⁺ puff generation due to the slow kinetics of Ca²⁺ binding by parvalbumin or due to the fact that a shorter peak amplitude is reached more quickly in these oocytes.

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Figure 4. Parvalbumin-induced Ca²⁺ puffs are characteristically different from Ca²⁺ puffs induced by injection of IP₃
A, single-line scan (~2.2 ms per line, 270 scans) of a Ca²⁺ puff induced by 50 µM parvalbumin. Bottom trace is a plot of the intensity ( $Delta$ F/F) along the line indicated by the arrow in the top panel (10 pixels wide). B, single-line scan of a Ca²⁺ puff induced by injecting an oocyte with IP₃ (30 nM, final). $Delta$ F/F is plotted in the bottom panel for a 10 pixel-wide line (indicated by the arrow). C, histograms of Ca²⁺ puff amplitude, decay time and rise time acquired in parvalbumin-injected oocytes. D, histograms of Ca²⁺ puff properties acquired in IP₃-injected oocytes. Arrows indicate the mean values for each histogram. Statistical significance: *** P < 0.0005, ** P < 0.001, * P < 0.05. The decay time is defined as the time elapsed from peak to one-half peak amplitude. The rise time is defined as the time elapsed from 10 to 90 % peak amplitude. All parameters were measured from raw, unprocessed images.

The Ca²⁺-binding protein calbindin D_28k does not evoke Ca²⁺ puffs

Calbindin D_28k is an EF-hand family member that contains four Ca²⁺-binding domains with a Ca²⁺ affinity of ~100 nM (Bredderman & Wasserman, 1974; Fullmer & Wasserman, 1987). In contrast to parvalbumin, calbindin D_28k rapidly binds Ca²⁺ since its affinity for Mg²⁺ is low (~1 mM) and its Ca²⁺-binding sites are largely unoccupied at resting concentrations of Mg²⁺. Given the Ca²⁺-stimulatory properties of parvalbumin and EGTA, we tested whether calbindin D_28k could also evoke Ca²⁺ puff activity. Oocytes were injected with purified calbindin D_28k protein and Ca²⁺ indicator dye, and imaged as described above. We found that injections of calbindin D_28k did not evoke significant Ca²⁺ puff activity at final concentrations of 0.5 nM (0 of 12 oocytes), 5 nM (0 of 22 oocytes, 50 nM (0 of 23 oocytes), 500 nM (0 of 23 oocytes) and 5 µM (0 of 19 oocytes). At 50 µM, 2 of 31 oocytes tested exhibited Ca²⁺ puffs. This level of Ca²⁺ activity was not significantly different from that observed in uninjected control oocytes (cf. Fig. 1C). To confirm that the injected concentrations of calbindin D_28k buffered cytosolic Ca²⁺, a subpopulation of calbindin D_28k-injected oocytes were subsequently injected with the non-metabolizable IP₃ analogue 1-( $alpha$ -glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate (GPIP₂, 300 nM final concentration) and imaged confocally. Calbindin D_28k significantly affected Ca²⁺ wave activity induced by exogenous injection of the IP₃ analogue. Specifically, we observed a slow elevation of cytosolic Ca²⁺ (Ca²⁺ tide) with no repetitive Ca²⁺ waves at either 7.5 or 1.25 µM calbindin D_28k (Fig. 5A and B). In contrast, oocytes injected with parvalbumin, and subsequently injected with GPIP₂, exhibited discrete Ca²⁺ release events (Fig. 6A and B) in addition to a Ca²⁺ tide. No GPIP₂-induced repetitive Ca²⁺ waves were observed in parvalbumin-injected oocytes. From these data we conclude that under our experimental conditions calbindin D_28k does not evoke Ca²⁺ puff activity.

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Figure 5. Calbindin D_28k inhibits repetitive Ca²⁺ waves, but does not evoke Ca²⁺ puffs
A and B, spatio-temporal stacks and representative images of IP₃-mediated Ca²⁺ activity in oocytes injected with calbindin D_28k (CB) at final concentrations of 7.5 µM (A) and 1.25 µM (B). C, repetitive Ca²⁺ activity in control oocytes injected with GPIP₂ (1 µM final) at 50 s. Confocal images were collected at 1 s intervals. Scale bars, 50 µm.

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Figure 6. Parvalbumin inhibits repetitive Ca²⁺ waves
A and B, IP₃-mediated Ca²⁺ activity in oocytes injected with parvalbumin (PV) at final concentrations of 50 µM (A) and 25 µM (B). Note the discrete sites of IP₃-mediated Ca²⁺ release in addition to a tide of Ca²⁺ release. C, control oocytes injected with GPIP₂ (1 µM final). Confocal images were collected at 1 s intervals. Scale bars, 50 µm.

BAPTA and Oregon Green 488 BAPTA-1 evoke Ca²⁺ puffs

Ca²⁺-dependent activation of the IP₃R is considered to be a faster process than Ca²⁺-dependent inactivation (Iino, 1990; Parker & Ivorra, 1990b; Bezprozvanny et al. 1991; Finch et al. 1991). Consequently, slow Ca²⁺ buffers may be more effective at evoking Ca²⁺ puffs than faster Ca²⁺ buffers, since they would have a reduced effect on Ca²⁺-dependent activation of the IP₃-bound IP₃R. Consistent with this hypothesis, parvalbumin and EGTA, which are relatively slow Ca²⁺ buffers, evoked Ca²⁺ puffs whereas calbindin D_28k, which is a rapid buffer, did not. To test whether the speed of Ca²⁺ binding was an important factor in eliciting Ca²⁺ puffs, we examined the effects of BAPTA on Ca²⁺ puff activity. BAPTA is a rapid Ca²⁺ buffer with a Ca²⁺ on-rate ~300 times faster than EGTA (Hellam & Podolsky, 1969; Tsien, 1980; Neher, 1986). We injected oocytes with a concentration of BAPTA (54 µM) that was adjusted to yield the same Ca²⁺-buffering capacity as 25 µM EGTA, where peak Ca²⁺ puff activity was previously observed (see Fig. 3B). An intracellular pH of 7.2 for Xenopus oocytes (Kang et al. 1998) was used for these calculations (Fabiato & Fabiato, 1979). All oocytes were also injected with Ca²⁺ indicator dye (12.5 µM Oregon Green 488 BAPTA-2) and imaged 30-60 min later. We found that BAPTA increased the percentage of oocytes exhibiting Ca²⁺ puffs (7 %, n = 210) as compared to control uninjected oocytes (2.4 %, n = 290; P < 0.005; Fig. 7A). The number of responding oocytes was not significantly different to the number evoked by the slow Ca²⁺ buffer EGTA, suggesting that the speed of Ca²⁺ binding is not a critical factor in evoking Ca²⁺ puffs. However, the mean Ca²⁺ puff frequency for BAPTA-injected oocytes (5.1 ± 1.3 events (100 s)^-1, n = 15) was approximately one-half of that observed for oocytes injected with EGTA (9.3 ± 0.44 events (100 s)^-1; Fig. 7B). This decrease in puff frequency indicates that rapid Ca²⁺ buffering is less efficient at controlling the frequency of Ca²⁺ puffs. Finally, since the Ca²⁺ indicator dye is also a rapid Ca²⁺ buffer, we tested whether Oregon Green 488 BAPTA-1 could itself evoke Ca²⁺ puffs. At a concentration of 48 µM, this Ca²⁺ indicator has an equivalent buffering capacity to 25 µM EGTA (Fabiato & Fabiato, 1979). We found that the percentage of responsive oocytes as well as the puff frequency were comparable to BAPTA-injected oocytes (Fig. 7A and B). Specifically, Oregon Green 488 BAPTA-1 evoked Ca²⁺ puff activity in 8.2 % of the oocytes tested (n = 290, P < 0.005). Of those responding, the mean frequency of puffs was 2 ± 0.3 events (100 s)^-1 (n = 24). It should be noted that all oocytes were also injected with the same basal concentration of Ca²⁺ indicator dye (12.5 µM Oregon Green 488 BAPTA-2). This concentration of Ca²⁺ indicator dye is an order of magnitude lower (12.5 µM) than the equivalent buffering capacity of 25 µM EGTA (i.e. 141 µM). It appears likely that the low level of puff activity observed in control oocytes is due to this basal concentration of Ca²⁺ indicator dye. Taken together, these data indicate that rapid Ca²⁺ buffers can evoke Ca²⁺ puffs, although less efficiently than slow Ca²⁺ buffers. We conclude that the speed of Ca²⁺ binding alone cannot account for the inability of calbindin D_28k to evoke Ca²⁺ puff activity.

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Figure 7. Mobile Ca²⁺ buffers evoke Ca²⁺ puffs
A and B, histograms of Ca²⁺ puff activity (A) and puff frequency (B), induced by EGTA (EG), BAPTA (BP), Oregon Green 488 BAPTA-1 (OG1) and Oregon Green 488 BAPTA-1 dextran (OGX). The controls (CN) are oocytes injected with Oregon Green 488 BAPTA-2 at 12.5 µM. *P < 0.005, compared to control (CN). C, plot of dye absorbance at 496 nm as a function of concentration for dextran-conjugated and non-conjugated Oregon Green 488 BAPTA-1. Each point represents the average of three measurements. Note that each dye exhibits identical absorption, demonstrating identical concentrations of Ca²⁺ indicator.

The dextran-conjugated substrate of Oregon Green 488 BAPTA-1 does not evoke Ca²⁺ puffs

The molecular weight of calbindin D_28k is approximately three times larger than that of parvalbumin (Bredderman & Wasserman, 1974; Pauls et al. 1996). Hence, the reduced mobility of calbindin D_28k may be a key factor in preventing it from evoking Ca²⁺ puffs. We tested the importance of buffer mobility by comparing Ca²⁺ puff activity in oocytes injected with Oregon Green 488 BAPTA-1 and its dextran conjugate (Oregon Green 488 BAPTA-1 dextran, 70 000 MW, Molecular Probes). The concentration of Oregon Green 488 BAPTA-1 dextran was adjusted to 48 µM to provide the same buffering capacity as 25 µM EGTA. The dextran conjugate was used after purification by centrifugation through a 30 000 MW cut-off filter to remove free dye (YM-30 device, Centricon-Millipore). To ensure that the two dyes were tested at equivalent concentrations, we measured the absorbance of each dye at 496 nm as a function of concentration (UV-1601 spectroscopy, Shimadzu) (Fig. 7C). When oocytes were injected with these dyes, we found that reducing the mobility of the Ca²⁺ buffer completely inhibited its ability to evoke Ca²⁺ puffs (Fig. 7A and B). Only 2.7 % (n = 290) of the oocytes injected with the dextran-conjugated Ca²⁺ buffer exhibited Ca²⁺ puff activity. Of those responding, the mean puff frequency was 2 ± 0.5 events (100 s)^-1 (n = 8). This level of activity was identical to that of control oocytes injected only with basal concentrations of Ca²⁺ indicator dye (12.5 µM, Oregon Green BAPTA-2). We conclude from these data that a high mobility of the Ca²⁺ buffer is a key factor in determining its efficacy to evoke Ca²⁺ puffs.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

In this paper, we demonstrate a novel function for parvalbumin in its ability to evoke Ca²⁺ puffs under resting, non-stimulated conditions. The ability of parvalbumin to evoke Ca²⁺ puffs depends on its Ca²⁺-binding properties and can be mimicked by EGTA, BAPTA or an equivalent concentration of Ca²⁺ indicator dye. However, Ca²⁺ buffering by itself does not appear to be sufficient to elicit Ca²⁺ activity, since neither the Ca²⁺-binding protein calbindin D_28k nor the dextran-conjugated Oregon Green 488 BAPTA-1 Ca²⁺ indicator dye could evoke Ca²⁺ puffs under our test conditions. The effect of parvalbumin was abolished by heparin, a known IP₃R blocker, suggesting that the increase in the Ca²⁺ puff activity is mediated by IP₃Rs bound with ligand. Measurements of the resting concentration of IP₃ are consistent with this model. Assuming an IP₃ affinity of 50 nM (Worley et al. 1987; Supattapone et al. 1988; Kaftan et al. 1997), a resting IP₃ concentration of 75 nM and a single, independent binding-site scheme (IP₃ + IP₃R JP8a IP₃-IP₃R), we estimate that over half of the IP₃R subunits are bound with IP₃. The probability of one subunit being bound with IP₃ is 0.6; the probability of two subunits being bound is 0.36 (i.e. 0.6 times 0.6); while the probability of three and four subunits being bound is 0.216 and 0.1296, respectively. Hence, in a cluster of 10 IP₃R subunit complexes, one to two ion channels are likely to have all four subunits bound, whereas three to four channels could have at least three subunits bound with ligand. Thus, there is potentially a sufficient number of IP₃Rs at resting concentrations of IP₃ to generate a Ca²⁺ puff. Our data also indicate that the basal concentration of IP₃ is not affected by injections of parvalbumin. It is unlikely that an increase in Ca²⁺ buffering produced undetectable local increases of IP₃ for the following reasons. First, our assay is sensitive enough to detect a 10 nM change in IP₃ concentration. In order to evoke Ca²⁺ puffs in oocytes, we required exogenous injections of 30-50 nM IP₃, which is clearly within the sensitivity of our measurement. Second, the ability of a buffer to stimulate an undetectable local increase in IP₃, for example by slowing IP₃ metabolism or releasing IP₃ from an uncharacterized compartment, should be independent of the mobility of the Ca²⁺ buffer. Hence, the inability of the dextran-conjugated Ca²⁺ buffer to evoke Ca²⁺ puffs argues strongly against a local increase in IP₃ being the underlying mechanism that accounts for the increase in Ca²⁺ puff activity.

Ca²⁺ puffs are thought to arise from the coordinated activation of a cluster of IP₃Rs (Parker et al. 1996; Bootman et al. 1997a; Horne & Meyer, 1997). We envisage a mechanism of action whereby parvalbumin increases the likelihood that a single IP₃R channel opening (Ca²⁺ blip) can recruit its neighbour(s) to open, subsequently synergizing and igniting the entire cluster of IP₃Rs (Ca²⁺ puff). We consider three potential mechanisms of action. First, binding of parvalbumin to Ca²⁺ could lower the local Ca²⁺ concentration, thereby reducing Ca²⁺-dependent inactivation of the IP₃R. This would result in more Ca²⁺ release, increasing the range over which neighbouring IP₃Rs can be recruited. However, when parvalbumin was injected into oocytes, we did not observe a decrease in the resting Ca²⁺ concentration. Since local changes in Ca²⁺ concentration may be undetectable, we also argued that such a mechanism of action would be dependent on the speed of Ca²⁺ buffering, since Ca²⁺-dependent activation is a faster process than Ca²⁺-dependent inactivation. We tested this hypothesis by determining whether we could induce Ca²⁺ puff activity using rapid Ca²⁺ buffers. We found that BAPTA evoked Ca²⁺ puffs in the same percentage of oocytes as the slower buffer EGTA. The latter, in turn, evoked Ca²⁺ puffs in fewer oocytes than the slowest Ca²⁺ buffer parvalbumin. Furthermore, of those oocytes exhibiting Ca²⁺ puffs, the frequency was lowest in BAPTA-injected oocytes. Thus, although we cannot entirely rule out the contribution of the speed of Ca²⁺ buffering in evoking puff activity, the on-rate of Ca²⁺ binding does not appear to be the critical factor.

Second, recent work indicates that Ca²⁺-dependent inactivation of the IP₃R may be mediated by calmodulin (Hirota et al. 1999; Michikawa et al. 1999; Missiaen et al. 1999). Calmodulin has been shown to bind to the IP₃R in a Ca²⁺-dependent manner (Yamada et al. 1995; Hirota et al. 1999). Parvalbumin could interfere with calmodulin binding to the receptor either by buffering Ca²⁺ or by binding directly to the receptor in competition with calmodulin. In support of a competitive binding mechanism, Ushio and co-workers have reported that parvalbumin directly interacts with a component of the sarcoplasmic reticulum (Ushio & Watabe, 1994). However, this explanation also appears unlikely since EGTA and BAPTA as well as the equivalent concentration of the Ca²⁺ indicator dye Oregon Green 488 BAPTA-1 can evoke Ca²⁺ activity. Finally, parvalbumin could facilitate the diffusion of Ca²⁺ away from the mouth of the ion channel, shuttling and releasing bound Ca²⁺ over a neighbouring site. To test this possibility, we hypothesized that higher molecular weight Ca²⁺ buffers should be less effective than more mobile, lower molecular weight buffers. We showed that the high molecular weight dextran-conjugated Ca²⁺ buffer did not evoke Ca²⁺ puffs, strongly demonstrating the importance of mobility in evoking Ca²⁺ puff activity. This mechanism of action provides an explanation for the inability of calbindin D_28k to effectively evoke Ca²⁺ puffs. Calbindin D_28k is larger than parvalbumin, decreasing its diffusional range. In addition, facilitated diffusion requires a Ca²⁺ buffer to be of sufficiently high affinity to shuttle the Ca²⁺ ion away from the open channel. Since the Ca²⁺ affinity of calbindin D_28k is lower than that of parvalbumin (~100 nM vs. 10 nM), the range over which calbindin D_28k can facilitate diffusion is correspondingly lower. Thus, we suggest that mobility, and hence facilitated diffusion, is a key factor in determining the efficacy of a Ca²⁺ buffer to evoke Ca²⁺ activity.

In summary, our data indicate that we need to consider two general mechanisms by which Ca²⁺ puffs can be generated: (1) the conventional mechanism, whereby sufficient spatial-temporal summation of IP₃-bound IP₃Rs is achieved by a hormone-induced increase in IP₃ (Fig. 8B; Bootman et al. 1997b; Sun et al. 1998; Thomas et al. 1998); and (2) a novel mechanism whereby the summation of events is affected by the expression of endogenous mobile cytosolic Ca²⁺ buffers such as parvalbumin (Fig. 8C). Interestingly, our data account for the experimental paradox in which Ca²⁺ activity in non-stimulated cells is rarely observed even though significant resting concentrations of IP₃ are measured (~75 nM). Specifically, we suggest that under resting conditions, the density of ligand-bound IP₃Rs in a cluster is too low to allow sufficient spatio-temporal summation to generate a Ca²⁺ puff (Fig. 8A). Consequently, ligand-bound IP₃Rs inactivate before neighbouring receptors can be recruited and sufficient Ca²⁺ is released to generate a detectable Ca²⁺ signal. The low level of Ca²⁺ puff activity that is observed in a few control oocytes is probably due to the mobile Ca²⁺ indicator dye used in imaging.

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Figure 8. Model of parvalbumin-evoked Ca²⁺ puffs
A cluster of IP₃Rs, from which a single Ca²⁺ puff can be evoked, is depicted in each panel. A, at resting IP₃ concentrations, the density of IP₃-bound receptors is too low to allow Ca²⁺ release from a single open receptor to activate neighbouring IP₃-bound receptors in the cluster. The green hemisphere depicts Ca²⁺ released from an open IP₃-bound IP₃R. Note that two of the IP₃-bound receptors are closed. Without the synergistic activation of neighbouring receptors, these elementary events are too short-lived to be detected by current imaging techniques. B, a hormone-induced increase in IP₃ levels increases the likelihood that neighbouring receptors will be bound by IP₃. This, in turn, increases the probability that bound receptors undergo spatio-temporal summation evoking a Ca²⁺ puff. C, low Ca²⁺ buffering by parvalbumin facilitates Ca²⁺ diffusion. This results in an increase in the distance through which Ca²⁺ can diffuse to stimulate neighbouring bound receptors, evoking a Ca²⁺ puff. Since fewer bound receptors contribute to Ca²⁺ puffs, the amplitude is expected to be lower in parvalbumin-induced puffs as compared to hormone-induced Ca²⁺ puffs. A larger diameter hemisphere indicates facilitated Ca²⁺ diffusion.

Previous investigations on the effects of parvalbumin on Ca²⁺ signalling have been undertaken in the presence of either an agonist- or voltage-induced increase in intracellular Ca²⁺ concentration (Gillis et al. 1982; Seamon & Kretsinger, 1983; Heizmann & Hunziker, 1991; Hou et al. 1991b; Chard et al. 1993; Jiang et al. 1996). Our study represents the first report of a CaBP increasing Ca²⁺ release, in the absence of extracellular stimulation. This effect was blocked by heparin, inhibited by mutation of the EF-hand Ca²⁺-binding domains of parvalbumin and could be mimicked by injecting the oocyte with low concentrations of mobile Ca²⁺ buffers. It is clear from these data that the initial expression of parvalbumin will have profound repercussions on the level of intracellular Ca²⁺ activity. Parvalbumin expression occurs in GABAergic neurons and in muscle cells at critical developmental stages (Celio & Heizmann, 1982; Berchtold, 1985, 1989; Kay et al. 1987; Muntener et al. 1987; Celio, 1988; Alcantara et al. 1996). Its expression has also been linked to several neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease (Heizmann & Braun, 1992; Krieger et al. 1996; Brady & Mufson, 1997). A parvalbumin isoform, oncomodulin, is preferentially expressed in tumour cells (Berchtold, 1989; Pauls et al. 1996). The ability of parvalbumin to increase Ca²⁺ puff activity also provides a potential mechanism for the generation of spontaneous Ca²⁺ sparks (the functional equivalent of Ca²⁺ puffs for the ryanodine receptor) in smooth muscle cells, which induce muscle relaxation and vasodilatation (Nelson et al. 1995). Clearly, cytosolic Ca²⁺ buffering by the Ca²⁺-binding protein parvalbumin can evoke intracellular Ca²⁺ puffs. Hence, the expression of parvalbumin must be considered as a potential agent that regulates Ca²⁺ excitability. We conclude that gene expression of certain CaBPs, in addition to hormones and voltage, plays a major role in controlling the dynamics of intracellular Ca²⁺ signalling.

REFERENCES

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Methods
Results
Discussion
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MUNTENER M., KASER, L., WEBER, J. & BERCHTOLD, M. W. (1995). Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA. Proceedings of the National Academy of Sciences of the USA 92, 6504-6508 [Abstract]

MUNTENER M., ROWLERSON, A. M., BERCHTOLD, M. W. & HEIZMANN, C. W. (1987). Changes in the concentration of the calcium-binding parvalbumin in cross-reinnervated rat muscles. Journal of Biological Chemistry 262, 465-469 [Abstract]

NEHER E. (1986). Concentration profiles of intracellular calcium in the presence of a diffusible chelator. Experimental Brain Research Series 14, 80-96

NELSON M. T., CHENG, H., RUBART, M., SANTANA, L. F., BONEV, A. D., KNOT, H. J. & LEDERER, W. J. (1995). Relaxation of arterial smooth muscle by calcium sparks. Science 270, 633-637 [Abstract]

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PARKER I., CHOI, J. & YAO, Y. (1996). Elementary events of InsP₃-induced Ca²⁺ liberation in Xenopus oocytes: hot spots, puffs and blips. Cell Calcium 20, 105-121 [Medline]

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PARKER I. & IVORRA, I. (1990b). Inhibition by Ca²⁺ of inositol trisphosphate-mediated Ca²⁺ liberation: A possible mechanism for oscillatory release of Ca²⁺. Proceedings of the National Academy of Sciences of the USA 87, 260-264 [Abstract]

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PAULS T. L., DURUSSEL, I., BERCHTOLD, M. W. & COX, J. A. (1994). Inactivation of individual Ca²⁺-binding sites in the paired EF-hand sites of parvalbumin reveals asymmetrical metal-binding properties. Biochemistry 33, 10393-10400 [Medline]

PUTNEY J. W. J. & BIRD, J. (1993). The inositol phosphate-calcium signaling system in nonexcitable cells. Endocrine Reviews 14, 610-631 [Medline]

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REINER A., MEDINA, L., FIGUEREDO-CARDENAS, G. & ANFINSON, S. (1995). Brainstem motoneuron pools that are selectively resistant in amyotrophic lateral sclerosis are preferentially enriched in parvalbumin: evidence from monkey brainstem for a calcium-mediated mechanism in sporadic ALS. Experimental Neurology 131, 239-250 [Medline]

SCHMALZING G., RUHL, K. & GLOOR, S. M. (1997). Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates. Proceedings of the National Academy of Sciences of the USA 94, 1136-1141 [Abstract/Full Text]

SEAMON K. B. & KRETSINGER, R. H. (1983) Calcium-modulated proteins In Metals in Biology. ed. SPIRO T. G., pp. 1-52. Wiley and Sons, New York

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STUHLFAUTH I. (1984). Calcium-binding protein, parvalbumin, is reduced in mutant mammalian muscle with abnormal contractile properties. Proceedings of the National Academy of the Sciences of the USA 81, 4814-4818

SUN X. P., CALLAMARAS, N., MARCHANT, J. S. & PARKER, I. (1998). A continuum of InsP₃-mediated elementary Ca²⁺ signalling events in Xenopus oocytes. Journal of Physiology 509, 67-80 [Abstract/Full Text]

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THOMAS D., LIPP, P., BERRIDGE, M. J. & BOOTMAN, M. D. (1998). Hormone-evoked elementary Ca²⁺ signals are not stereotypic, but reflect activation of different size channel clusters and variable recruitment of channels within a cluster. Journal of Biological Chemistry 273, 27130-27136 [Abstract/Full Text]

TSIEN R. Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures. Biochemistry 19, 2396-2404 [Medline]

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MUNTENER M., KASER, L., WEBER, J. & BERCHTOLD, M. W. (1995). Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA. Proceedings of the National Academy of Sciences of the USA 92, 6504-6508	[Abstract]
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NEHER E. (1986). Concentration profiles of intracellular calcium in the presence of a diffusible chelator. Experimental Brain Research Series 14, 80-96
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PARKER I. & IVORRA, I. (1990a). Localized all-or-none calcium liberation by inositol trisphosphate. Science 250, 977-979	[Medline]
PARKER I. & IVORRA, I. (1990b). Inhibition by Ca²⁺ of inositol trisphosphate-mediated Ca²⁺ liberation: A possible mechanism for oscillatory release of Ca²⁺. Proceedings of the National Academy of Sciences of the USA 87, 260-264	[Abstract]
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PUTNEY J. W. J. & BIRD, J. (1993). The inositol phosphate-calcium signaling system in nonexcitable cells. Endocrine Reviews 14, 610-631	[Medline]
REBER B. F. X. & SCHINDELHOLZ, B. (1996). Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites. Pflügers Archiv 432, 893-903	[Medline]
REINER A., MEDINA, L., FIGUEREDO-CARDENAS, G. & ANFINSON, S. (1995). Brainstem motoneuron pools that are selectively resistant in amyotrophic lateral sclerosis are preferentially enriched in parvalbumin: evidence from monkey brainstem for a calcium-mediated mechanism in sporadic ALS. Experimental Neurology 131, 239-250	[Medline]
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SEAMON K. B. & KRETSINGER, R. H. (1983) Calcium-modulated proteins In Metals in Biology. ed. SPIRO T. G., pp. 1-52. Wiley and Sons, New York
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STUHLFAUTH I. (1984). Calcium-binding protein, parvalbumin, is reduced in mutant mammalian muscle with abnormal contractile properties. Proceedings of the National Academy of the Sciences of the USA 81, 4814-4818
SUN X. P., CALLAMARAS, N., MARCHANT, J. S. & PARKER, I. (1998). A continuum of InsP₃-mediated elementary Ca²⁺ signalling events in Xenopus oocytes. Journal of Physiology 509, 67-80	[Abstract/Full Text]
SUPATTAPONE S., WORLEY, P. F., BARABAN, J. M. & SNYDER, S. H. (1988). Solubilization, purification, and characterization of an inositol trisphosphate receptor. Journal of Biological Chemistry 263, 1530-1534	[Abstract]
THOMAS D., LIPP, P., BERRIDGE, M. J. & BOOTMAN, M. D. (1998). Hormone-evoked elementary Ca²⁺ signals are not stereotypic, but reflect activation of different size channel clusters and variable recruitment of channels within a cluster. Journal of Biological Chemistry 273, 27130-27136	[Abstract/Full Text]
TSIEN R. Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures. Biochemistry 19, 2396-2404	[Medline]
UNDERWOOD R., GREELEY, R., GLENNON, E., MENACHERY, A., BRALEY, L. & WILLIAMS, G. (1988). Mass determination of polyphosphoinositides and inositol triphosphate in rat adrenal glomerulosa cells with a microspectrophotometric method. Endocrinology 123, 211-219	[Abstract]
USHIO H. & WATABE, S. (1994). Carp parvalbumin binds to and directly interacts with the sarcoplasmic reticulum for Ca²⁺ translocation. Biochemical and Biophysical Research Communications 199, 56-62	[Medline]
VOLPE P., ALDERSON-LANG, B. H. & NICKOLS, G. A. (1990). Regulation of inositol 1,4,5-trisphosphate-induced Ca²⁺ release. I. Effect of Mg²⁺. American Journal of Physiology 258, C1077-1085	[Medline]
WATRAS J., BEZPROZVANNY, I. & EHRLICH, B. E. (1991). Inositol 1,4,5-trisphosphate-gated channels in cerebellum: presence of multiple conductance states. Journal of Neuroscience 11, 3239-3245	[Abstract]
WORLEY P. F., BARABAN, J. M., SUPATTAPONE, S., WILSON, V. S. & SNYDER, S. H. (1987). Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium. Journal of Biological Chemistry 262, 12132-12136	[Abstract]
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