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J. Biol. Chem., Vol. 277, Issue 23, 20256-20263, June 7, 2002

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Carboxyl-terminal Peptide of -Amyloid Precursor Protein Blocks Inositol 1,4,5-Trisphosphate-sensitive Ca²⁺ Release in Xenopus laevis Oocytes^*

Joung-Hun Kim^§, Jong-Cheol Rah^¶, Scott P. Fraser, Keun-A Chang^¶, Mustafa B. A. Djamgoz, and Yoo-Hun Suh^¶

From the  Neurobiology Group, Department of Biology, Sir Alexander Fleming Bldg., Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom and the ^¶ Department of Pharmacology, College of Medicine, National Creative Research Initiative Centre for Alzheimer's Dementia, and Neuroscience Research Institute, MRC, Seoul National University, Seoul 110-799, South Korea

Received for publication, August 29, 2001, and in revised form, February 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The effects of Alzheimer's disease-related amyloidogenic peptides on inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ mobilization were examined in Xenopus laevis oocytes. Intracellular Ca²⁺ was monitored by electrophysiological measurement of the endogenous Ca²⁺-activated Cl current. Application of a hyperpolarizing pulse released intracellular Ca²⁺ in oocytes primed by pre-injection of a non-metabolizable inositol 1,4,5-trisphosphate analogue. The carboxyl terminus of the amyloid precursor protein inhibited inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca²⁺ release in a dose-dependent manner. Equimolar -amyloid peptides A_1-40 or A_1-42 had no effect, and whereas a truncated carboxyl terminus lacking the A domain was equipotent to the full-length one, a carboxyl terminus fragment lacking the NPTY sequence was less effective than the full-length fragment. The inhibition induced by the carboxyl terminus was not associated with the block of the Ca²⁺-dependent Cl channel itself or compromised Ca²⁺ influx. We conclude that the carboxyl terminus of the amyloid precursor protein inhibits inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release and could thus disrupt Ca²⁺ homeostasis and that the carboxyl terminus is much more effective than the -amyloid fragments used. By perturbing the coupling of inositol 1,4,5-trisphosphate and Ca²⁺ release, the carboxyl terminus of the amyloid precursor protein can potentially be involved in inducing the neural toxicity characteristic of Alzheimer's disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Alzheimer's disease (AD)¹ is a progressive and fatal neurodegenerative disease characterized by amyloid plaques and neurofibrillary tangles (for a review, see Ref. 1). These plaques are associated with degenerating neuronal processes and consist primarily of fibrillar aggregates of -amyloid protein (A). Although A fragments are the main component of amyloid plaques and have been shown to damage or kill cultured neurones (for a review, see Ref. 2), A may not be the sole neurotoxic component of the amyloid precursor protein (APP). For example, A peptide deposition has been observed without accompanying neurodegeneration (3-5). Another amyloidogenic fragment, the carboxyl terminus (CT) of APP, which is composed of 99-105 amino acid residues containing the complete A sequence, also appears to be toxic to neurones (6, 7). CT has been found not only in presynaptic terminals of rodent entorhinal neurones (8) but also in paired helical filaments (9), in senile plaques (10), in microvessels (11-13), in choroid plexus from human brain in AD, in the white matter of Down's syndrome brains (14), and in both cytosol and media of lymphoblastoid cells obtained from patients with early- or late-onset familial AD (15) and Down's syndrome (16). A transgenic mouse model expressing CT exhibited neuropathology very similar in many respects to that of AD, especially as regards age-dependent neuronal and synaptic degeneration (17) and spatial-learning deficits and electrophysiological alterations that suggested impairment of synaptic plasticity (18). A 31-amino acid carboxyl-terminal fragment of APP (CT31) generated from CT by caspase-9 cleavage was reported to have a pro-apoptotic effect and to mainly contribute to the CT toxicity in neuronal cells. Both CT31 and activated caspase-9 were present in the brains of AD patients but not in control brains (19). These findings together indicate that CTs themselves may also contribute to the neurodegenerative processes in vivo not only as a precursor to make A. Earlier studies showed that A-bearing CTs were released from several different cells and/or were more easily released from the damaged neurons into the media or extracellular fluid (20-22). Recent data has shown the presence of CT in the nuclei (23) and the transcriptively active complex of CT with Fe65 and histone acetyltransferase, Tip60 (24). These findings suggest that CT plays important roles in delayed neurodegeneration via transcriptional regulation. Our previous study also demonstrated that CT fragments without A and the transmembrane domain may also exert toxicity in nerve growth factor-differentiated PC12 cells (25). CT can also contribute to stimulation of inflammatory processes linked to delayed neurodegeneration in AD (26-28).

A and CT peptides have been shown to damage cultured neurons by a mechanism involving disruption of Ca²⁺ homeostasis (29, 30). However, the potential role of amyloidogenic fragments, including A, in IP₃ production and/or intracellular Ca²⁺ mobilization has not been clear.

Elucidating the possible effects of A or CT peptides on IP₃ signaling has been hampered by the intracellular location of IP₃ receptors. Furthermore, limitations concerning specific inhibitors or activators of the IP₃ receptors, not affecting ryanodine receptors, have also made it difficult to investigate A effects on the IP₃ pathway (31). To circumvent these difficulties, we have used Xenopus laevis oocytes as a model system. The large size of Xenopus oocytes facilitated precise intracellular application of IP₃ and APP fragments. Moreover, the Ca²⁺ response of Xenopus oocytes was not complicated by ryanodine receptors, which are absent in the oocytes (32). Another advantage of this model system is that injection of IP₃ into Xenopus oocytes elevates the cytosolic Ca²⁺ level and activates Ca²⁺-dependent Cl currents (33), which can be used as a real-time indicator of cytosolic Ca²⁺ concentration (34, 35). In addition, Yao and Parker (36) showed that application of a hyperpolarizing pulse to Xenopus oocytes pre-injected with an enzyme-resistant IP₃ analogue, D-3-deoxy-3-fluoro-myo-inositol 1,4,5-trisphosphate (3-F-IP₃), induced a current that represented Ca²⁺ release from IP₃-sensitive stores. We have adopted this protocol to study the effect of amyloidogenic peptides on the IP₃ pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Recombinant CT105 and A-- Recombinant CT105 was prepared on the basis of human APP770 cDNA as described previously (37). Briefly, the expression plasmid pCS-CT105 was constructed by ligating the 704-bp BglII-ClaI fragment excised from pSP65-APP770 into ptrpSF9, digested with BglII and SmaI, treated with calf intestinal alkaline phosphatase, and transformed into Escherichia coli XL1-Blue. CT105 peptide (M_r 14,242) was purified by a combination of urea solubilization and ion exchange chromatography and then subjected to dialysis against 10 mM Tris-HCl (pH 7.4) followed by lyophilization. To rule out the effect of possible co-purified contaminating factors, groups incubated with polymixin B sulfate were compared with non-treated ones. We confirmed that these are not significantly different. A_1-42 was purchased from Sigma-Aldrich Co. Ltd. (Dorset, UK). A_1-40, A_1-42, and CT were dissolved in distilled water to make 100 µM stock solutions and incubated at 37 °C for 6 days.

Construction of Truncated CT Peptides-- To determine which region is the active domain responsible for the effect on IP₃ signaling of CT, we have generated truncated carboxyl-terminal fragments of APP. Because A, the transmembrane portion, and the NPTY sequence in the cytoplasmic tail are believed to be important in mediating toxic effects of CT peptide, constructs without these domains were generated using the PCR-amplified strategy and the GST fusion protein strategy as described earlier (25). Briefly, they were cloned from pSP65-APP770 by PCR using oligonucleotides complementary to corresponding position (28). For the construction of plasmids encoding GST fusion proteins, BamHI/XhoI fragment of the PCR-generated fragments was cloned into the BamHI/XhoI site of pGEX 4T-1. We have prepared the following constructs: GST-CT 46 (649-695) without A and transmembrane (TM) domain (CTdA/TM) and GST-CT 86 (597-683) without NPTY domain (CTdNPTY). These final PCR constructs were confirmed by automated DNA sequencing. These recombinant GST-CTs of APP were purified with a combination of urea solubilization and ion exchange chromatography.

Oocyte Preparation and Solutions-- X. laevis oocytes were prepared and defolliculated as described previously (38). Defolliculated oocytes were stored in incubation medium at 19 °C for several days. Composition of the incubation medium was (in millimolar): NaCl, 88.0; KCl, 1.0; CaCl₂, 0.41; NaHCO₃, 2.5; MgSO₄, 0.82; and Ca(NO₃)₂, 0.33, penicillin and streptomycin, 10 µg/ml each; gentamicin sulfate, 0.1 mg/ml; pH 7.5. Unless otherwise noted, oocytes were continually perfused with Ringer's solution at room temperature (18-20 °C) during recording. In the case of thapsigargin pretreatment, oocytes were incubated in Ca²⁺-free medium containing 1 µM thapsigargin overnight at 19 °C.

Composition of the normal Ringer's solution was (millimolar): NaCl, 120; KCl, 2; CaCl₂, 1.8; HEPES, 5; pH 7.3. Ca²⁺-free medium was made by omitting CaCl₂ and adding 5 mM MgCl₂ together with 1 mM EGTA (36). Most chemicals were purchased from Sigma-Aldrich Company Ltd. (Dorset, UK). 3-F-IP₃, purchased from CN Biosciences UK (Nottingham, UK), was dissolved in distilled water at 35 µM. 3-F-IP₃ is about equipotent to IP₃ in liberating intracellular Ca²⁺ but cannot be converted to inositol 1,3,4,5-tetrakisphosphate by the action of 3-kinases and thus stimulates IP₃ receptors continuously (39). Thapsigargin (Alomone Labs Ltd., Jerusalem, Israel) was dissolved in dimethyl sulfoxide (Me₂SO). The final concentration of Me₂SO in the Ca²⁺-free medium was 0.1%. The dissolved chemical and peptides were kept at 20 °C until use.

Microinjection-- Pulled injection pipette3-00-2s (03-G/XL, Drummond, Broomal, PA; tip diameters, 10-15 µm) were back-filled with the 3-F-IP₃ solutions and attached to an oocyte injector (Drummond). 46 nl of solution was injected into each oocyte. Injected oocytes were kept for about 30 min in an incubator at 19 °C, except for experiments on transient current responses. The latter were induced directly by 3-F-IP₃ injections, under voltage clamp, using a pneumatic pico-pump (WPI, Sarasota, FL). A holding potential of 40 mV was used in these experiments.

In the main body of experiments, A_1-40, A_1-42, full-length CT or the truncated fragments were injected into oocytes which had been "primed" previously for 30 min with 3-F-IP₃ (at 150 nM intracellular concentration). Injection of the peptides was achieved either with the oocyte injector (Drummond) or with the pico-pump, dependent on the experimental set up. For experiments using the pico-pump, the volume of the injected peptide solution was calibrated before the start of the experiments. For a pre-set pressure (20 p.s.i.), the duration was adjusted to give the same ejected volume of liquid (46 nl), measured under a dissection microscope. The duration was determined separately for each injection pipette. The concentrations of peptides and 3-F-IP₃ injected into oocytes are given in the text as the final, intracellular ones. These were as follows: 0.22, 0.44, 0.88, 1.3, 1.8, 2.2, and 3.0 µM for CT; 0.44, 0.88, 2.2, and 3.0 µM for A_1-40 and A_1-42; and 1.2 mM for the various truncated fragments.

Electrophysiology-- Conventional two-electrode methodologies were performed with a voltage-clamp amplifier (GeneClamp 500B, Axon Instruments, Foster City, CA). Resistances of the voltage-measuring electrodes filled with 2.5 M KCl were in the range 1-3 M, whereas current-measuring electrodes had resistances of 0.4-1 M. Stimulation and data acquisition were controlled by pClamp 6.02 software (Axon Instruments) via a Digidata 1200 interface (Axon Instruments). Recorded data were stored on an IBM computer and analyzed in pClamp 6.02 package. Two types of membrane currents were recorded: 1) Fast, transient currents induced directly by 3-F-IP₃ injection. For recording the transient current (I_t) induced directly by 3-F-IP₃ itself, the compound was injected into oocytes under voltage clamp (40 mV), and the resulting currents were monitored for ~20 min. 2) Late currents induced by membrane hyperpolarization in oocytes pre-injected with 3-F-IP₃. The delayed membrane currents (T_in) were recorded as follows: (i) An oocyte pre-injected with 3-F-IP₃ was incubated for 30 min and transferred to the recording chamber. (ii) The oocyte was voltage-clamped at a holding potential of 0 mV to keep the driving force of Ca²⁺ influx at a low level. (iii) A hyperpolarizing pulse was applied from 0 mV to 110 mV for 4.5 s, and the current response was recorded. The current response recorded before the peptide injection was used as the control. (iv) A peptide was injected with the pico-pump or oocyte injector. (v) The same hyperpolarizing pulse was applied to the oocyte 10 min following peptide injection. The resulting response was compared with its own pre-injection control. The percentage residual current was expressed as [(post-injection amplitude/pre-injection amplitude) × 100].

Data Analysis-- All data have been presented as means ± S.E. of the means (S.E.). The number of measurements given (n) represents different oocytes; the number of batches, i.e. toads used (N) for a given experiment, is also indicated. Data were analyzed using a statistics package, SAS/6.03 (SAS Institute, Cary, NC). Effect of a given peptide on current amplitude was analyzed by one-way ANOVA, after all the percentage data were transformed to logarithmic values. A Tukey test was used for multiple comparisons. Wilcoxon test (non-parametric) was used for comparison of two groups. Correlation coefficients were determined by non-parametric Spearman test. Statistical significance was considered at the following levels: p < 0.05 (), p < 0.01 (*), p < 0.001 (**), p < 0.0001 (***), and p < 0.00001 (****).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Control Experiments

3-F-IP₃-induced Transient Membrane Currents-- Injection of 3-F-IP₃ (1 µM) into oocytes held at 40 mV caused a biphasic inward current: A fast, transient component (I_t), followed by a slow phase with typical oscillations (Fig. 1A). This response pattern was similar to those induced by IP₃ or 3-F-IP₃ in previous studies on Xenopus oocytes (36, 40, 41). This confirmed that the IP₃ system was functioning normally.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Left panel, A, membrane current recorded from an oocyte (voltage-clamped at 40 mV) in response to injection of 1 µM 3-F-IP3. The injection of 3-F-IP3 activates a large transient inward current (It) followed by a long-lasting secondary inward current with some oscillations. The arrowhead indicates the injection point of 3-F-IP3. B, hump current (Tin) evoked by a hyperpolarizing pulse applied to oocytes previously injected with 3-F-IP3 (150 nM). The oocyte was loaded with 3-F-IP3 about 30 min before applying the voltage pulse (40 to 110 mV; 4.5-s duration). C, similar protocol (as in B) applied to control, non-injected oocyte. No hump current is generated. Currents were not leak-subtracted. Right panel, effect of extracellular Ca2+ on Tin. D, the control current response of a 3-F-IP3-injected oocyte bathed in normal Ringer's solution. E, oocyte bathed in Ca2+-free Ringer's solution. F, recovery on returning to normal Ringer's solution. The recordings were obtained from the same oocyte. The scale bar in A applies to part A, whereas the scale bar in part B refers to all other parts of the figure.

Hyperpolarization-induced Membrane Currents in 3-F-IP₃-injected Oocytes-- Oocytes injected with 3-F-IP₃ (150 nM) were incubated for a further 30 min and then a hyperpolarizing pulse (0 to 110 mV) was applied under voltage clamp. This generated a slow "hump-like" inward current (T_in, Parker and Miledi (42)) (Fig. 1B). Water-injected control oocytes showed only leakage currents without T_in (Fig. 1C). The reversal potential of T_in was about 22 mV (n = 3), similar to the value reported previously for the endogenous Ca²⁺-dependent Cl current of Xenopus oocytes (e.g. Ref. 34).

Effect of Enhanced Depletion of Ca²⁺ Stores on T_in-- A high level of 3-F-IP₃ (1.5 µM) was used to test whether T_in could be altered by enhanced depletion of Ca²⁺ stores. The mean amplitudes of the hump currents induced by 1.5 µM and 150 nM 3-F-IP₃ were indistinguishable: 962 ± 192 nA (n = 14/n = 3) versus 1153 ± 233 nA (n = 15/n = 3; p > 0.1), respectively. This was not due to 150 nM 3-F-IP₃ saturating the Ca²⁺ release pathway. This was confirmed by comparing the amplitudes of I_t generated directly by the IP₃ analogue injection: The currents induced by 150 nM and 1.5 µM 3-F-IP₃ were 112.6 ± 32.2 and 279.3 ± 61.9 nA, respectively (n = 7/n = 2 for both; p < 0.05). These results suggested that the amplitude of T_in was not affected by possible enhanced depletion of the Ca²⁺ stores.

Ca²⁺ dependence of T_in-- We examined whether T_in depended on extracellular Ca²⁺. T_in was abolished in the Ca²⁺-free/EGTA Ringer's solution (Fig. 1, D versus E). This effect was reversible, with the current recovered in normal Ringer's solution containing 1.8 mM Ca²⁺ (Fig. 1F). We also tested whether T_in could be mediated exclusively by Ca²⁺ influx. Thapsigargin (1 µM) was used to deplete Ca²⁺ stores and set up capacitative Ca²⁺ entry (43). No T_in current could be observed in such oocytes using the standard protocol (data not shown). This confirmed, under our experimental conditions, T_in was not simply generated by Ca²⁺ entering from outside.

These characteristics taken together are consistent with the T_in current representing a Cl current activated by Ca²⁺ released from IP₃-sensitive stores by the triggering action of hyperpolarization-induced Ca²⁺ influx, as in the study of Yao and Parker (36).

Inhibitory Effect of CT on T_in

Following a control (pre-injection) recording of T_in in oocytes primed with 3-F-IP₃, water (as vehicle control) or an amyloidogenic fragment of APP (A_1-40, A_1-42, or CT) was applied intracellularly. Experiments, in which the oocytes were injected with distilled water alone, showed that T_in was stable and that the post-injection amplitudes were not much different from those of pre-injection controls (residual current = 96.5 ± 4.8%; n = 18/n = 4; p > 0.05; Figs. 2A and 3A). Application of either A_1-40 or A_1-42 (0.22-3.0 µM for both) had no significant effect on T_in (n = 8 for each; Figs. 2B, 2C, and 3A). In contrast, injection of CT peptide was very effective in suppressing T_in (Figs. 2D and 3A). The inhibitory effect of CT peptide on T_in was dose-dependent (Fig. 4). The concentration of CT peptide that induced half-maximal inhibition of T_in was about 0.4 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effects of amyloidogenic fragments on Tin. Traces in the left panel show control recordings of Tin before injection. Corresponding traces on the right show the current responses after injection in each case. A, water injection as a vehicle control. B, 3 µM A1-40; C, 3 µM A1-42; D, 3 µM CT. The same volume (46 nl) of sterile distilled water or peptide solution was injected into each oocyte. Other experimental conditions were as in Fig. 1B.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   A, normalized mean amplitudes of residual Tin after injection of water or 2.2 µM amyloidogenic fragments. Residual currents (%) were calculated by dividing the peak amplitude of Tin after injection with that of the respective control Tin (i.e. (post-injection Tin × 100)/(pre-injection Tin)). One way-ANOVA and a Tukey test were performed for statistical comparisons with the water-injected control (***, p < 0.0001). The data for each condition represent the mean of eight oocytes; the value for water control was obtained from 12 oocytes. Error bars denote S.E. B, normalized mean amplitudes of residual Tin after injection of water, GST, 1.22 µM CT, or equimolar truncated CT peptides. Residual currents were determined as described above. Student's t test was performed for comparison with GST control (*, p < 0.01; CTdNPTY (+) indicates p < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for the effects of CT (filled circle), A1-40 (filled square), and A1-42 (open circle) on Tin. Residual currents (%) were calculated as stated in Fig. 3 legend and under "Experimental Procedures." One way-ANOVA and then a Tukey test were performed for statistical comparisons to water-injected control. Each point is the average of measurements from 8-12 oocytes. The error bars denote S.E.

We also investigated if CT would inhibit I_t induced directly by 3-F-IP₃ injection. 3-F-IP₃ (1 µM) typically evoked peak inward currents of 50-350 nA. There was no statistically significant effect on the peak inward current when 1.3 µM CT (a concentration that consistently inhibited T_in) was co-injected with 3-F-IP₃ (n = 13/n = 3; p > 0.1). In addition, the reduction of T_in was not overcome by 10- or 100-fold higher concentration of 3-F-IP₃ (i.e. 1.5 and 15 µM, respectively; data not shown). In addition, we tested whether IP₃ re-injection could evoke the current following the initial block by CT. We could not find any recovery of T_in in this condition (data not shown).

Comparison of Inhibitory Effect of CT and the Truncated CT Peptides on T_in-- To explore the active part of the CT domain responsible for its inhibitory effects on T_in, we made truncated CT peptides, CTdNPTY and CTdA/TM. Water, 1.2 µM GST, CT, or each GST-conjugated, truncated CT peptide was applied intracellularly, after checking T_in in oocytes primed with 3-F-IP₃. CTdNPTY suppressed T_in less effectively than full-length CT injection (residual current = 57.2 ± 10.3%, n = 7; p < 0.05; Fig. 3B), whereas CTdA/TM showed no significant difference (residual current = 19.3 ± 10.3%, n = 7; Fig. 3B) at the same concentration. The control oocytes injected with GST or water showed stable T_in (residual current of GST = 101.0 ± 15.5%, n = 7; p > 0.05; residual current of water = 96.5 ± 4.8%, n = 18; p > 0.05; Fig. 3A). These results show that the YENPTY domain in the cytoplasmic tail seems to have a more crucial role in inhibiting IP₃ signaling rather than the A or TM domains.

Lack of Effect of CT on Capacitative Ca²⁺ Entry or Ca²⁺-dependent Cl Channels-- To eliminate the possibility that CT could compromise capacitative Ca²⁺ entry, oocytes that had been pre-treated with thapsigargin overnight (in the absence of extracellular Ca²⁺), to set up capacitative Ca²⁺ entry (44), were injected with 1.3 µM CT. Current responses to hyperpolarizing pulses were 646 ± 142 nA before, compared with 590 ± 157 nA following CT injection (p = 0.18, n = 11/n = 3; Fig. 5). In addition, thapsigargin treatment is known to lengthen the time-to-peak of T_in (41). We therefore measured the time-to-peak of T_in before and following 1.3 µM CT injection (1245 ± 196 ms versus 1134 ± 150 ms, respectively). The values were not significantly different (p = 0.19; n = 12). Together, these results are consistent with CT having no significant effect on capacitative Ca²⁺ entry.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Lack of effect of CT on capacitative Ca2+ entry. Oocytes were incubated in Ca2+-free medium containing 1 µM thapsigargin overnight before recording. A hyperpolarizing pulse (40 to 110 mV for 4.5 s) was applied. Currents were recorded before (A) and following (B) CT peptide injection. The traces were obtained from different oocytes.

To elucidate whether the inhibitory effect of CT involved directly the Ca²⁺-activated Cl channels, we compared the decay time courses of T_in before and after CT injection in oocytes in which CT reduced, but did not abolish, the peak T_in current (see later). Current decay could be fitted with a single exponential of which the time constant () did not change significantly following CT injection ( = 1024 ± 265 versus 964 ± 156 ms, before and after CT, respectively, n = 16/n = 4; p > 0.4; Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Decay time courses of Ca2+-activated Cl channels. These were not altered by CT injection. Representative traces of Tin evoked by hyperpolarization from 0 mV to 110 mV in 3-F-IP3-injected oocytes, recorded before (A) and following (B) CT injection. The traces are from different oocytes. The straight lines represent the exponential best fits to the current decays, with time constants () as indicated.

In conclusion, the inhibitory effect of CT on Ca²⁺ release was most likely due to suppression of IP₃-sensitive Ca²⁺ release from internal stores, rather than any effect on capacitative Ca²⁺ entry or Ca²⁺-activated Cl current.

Relation of Time to Peak of T_in and Inhibitory Effect of CT-- We explored the relationship between the time-to-peak of T_in (measured before CT injection) and the residual peak current recorded after CT injection expressed as a percentage of the original amplitude. Fig. 7 shows that there was a strong inverse correlation between the time-to-peak prior to CT injection and the residual current (correlation coefficient = 0.78; p < 0.001). This suggested that oocytes with slower T_in were more sensitive to CT. In other words, when Ca²⁺ influx (derived from the membrane hyperpolarization) needed to trigger T_in was greater, as indicated by the time-to-peak of T_in, the suppressing effect of CT was bigger.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Correlation of residual current amplitudes (normalized, %; as described in the legend to Fig. 3) and time-to-peak (log scale). The inhibitory effect of CT on Tin was enhanced in oocytes displaying longer time-to-peak before CT peptide injection. A non-parametric Spearson test showed that there is a strong inverse correlation between the two data sets (correlation coefficient = 0.78; p < 0.001).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We investigated the effects of amyloidogenic fragments of APP on the IP₃ pathway in X. laevis oocytes using the Ca²⁺-activated Cl (T_in) current as an indicator of [Ca²⁺]_i. The main conclusion is that CT peptide, but not the A fragments, inhibits Ca²⁺ release from intracellular Ca²⁺ stores when activated by an IP₃ analogue and triggered by hyperpolarization-induced Ca²⁺ influx.

T_in required the presence of extracellular Ca²⁺ to be triggered, because it was abolished in Ca²⁺-free medium (Fig. 1, E and F). If T_in were to be mediated exclusively by Ca²⁺ entry, however, it should have persisted in oocytes when capacitative Ca²⁺ entry was induced by use of thapsigargin (43). The absence of T_in in thapsigargin-treated oocytes suggested that the current was not dependent upon Ca²⁺ influx alone. It was concluded, therefore, that entry of Ca²⁺ induced by membrane hyperpolarization triggered T_in, as a consequence of Ca²⁺ release from IP₃-sensitive Ca²⁺ stores in continuous presence of IP₃. This is consistent with the results of Yao and Parker (36). It should be noted, however, that under conditions associated with more "complex" second-messenger associated events (e.g. 5-hydroxytryptamine receptor activation (36)), there is evidence that T_in can be activated by interaction of Ca²⁺ influx and Cl channels, in a pathway that bypasses the need for intracellular Ca²⁺ release. However, under our experimental conditions, this was clearly not the case.

Enhanced depletion of the Ca²⁺ store obtained by high (1.5 µM) concentration of 3-F-IP₃ did not result in any significant increase of T_in amplitude. This ruled out the possibility that T_in was a simple delayed release of Ca²⁺ from incompletely depleted Ca²⁺ stores. However, it is possible that the IP₃-dependent Ca²⁺ release could involve a refilling mechanism occurring after Ca²⁺ store depletion.

Control experiments, in which oocytes were injected with distilled water alone, showed that T_in was stable and that the post-injection amplitude was not much different from the pre-injection amplitude (Figs. 3A and 4). Intracellular application of the CT peptide strongly attenuated T_in, whereas A_1-40 and A_1-42 had no significant effect under identical experimental conditions (Figs. 3, B-D, and 4A). In addition, the reduction of T_in was not overcome by 10- or 100- fold higher concentration of 3-F-IP₃ (i.e. 1.5 and 15 µM, respectively, data not shown). In addition, we tested whether IP₃ re-injection could evoke the current following the initial block by CT. We could not find any recovery of T_in in this condition (data not shown). These experiments indicate that CT inhibits T_in by altering the IP₃-induced receptor-signaling pathway rather than simply by blocking IP₃ receptor directly.

The deletion studies showed that YENPTY sequence, which is known to have a role in intracellular sorting (24), rather than the A or TM domains, may at least in part, be responsible for the attenuation of IP3 induced signal (Fig. 3B).

Interestingly, injection of the CT peptide had no effect on the size of I_t. At present, the reasons for this are unclear, because both I_t and T_in are dependent on IP₃. However, the two events are distinct in that (i) I_t is an immediate event following IP₃ injection, whereas T_in takes many minutes to develop and (ii) I_t, unlike T_in, is not dependent upon extracellular Ca²⁺ (45). Thus, possible differences in the intracellular events controlling these two discrete events may explain their distinct sensitivity to CT.

The inhibitory effect of CT on T_in was concentration-dependent, with the half-maximal effect occurring at about 0.4 µM (Fig. 4). This is in the range of CT concentrations found previously to be toxic to oocytes (46) and to a variety of mammalian cells, including neurones (7).

Several possible mechanisms were tested to elucidate the one or more steps involved in the inhibitory effect of CT on T_in. In particular, the possible effects of CT on 1) capacitative Ca²⁺ entry, 2) the Ca²⁺-activated Cl channel, or 3) the cytoplasmic Ca²⁺ level, were evaluated.

Compromised Ca²⁺ entry, preventing Ca²⁺ release from intracellular stores, was unlikely to be involved, because capacitative Ca²⁺ entry induced by pre-treatment of thapsigargin did not change significantly after CT injection (Fig. 5) and CT had no effect on the length of the time-to-peak of T_in. This is in agreement with recent work by Yoo et al. (47), who found that neither A_1-42 nor full-length APP had any effect on capacitative Ca²⁺ entry in Chinese hamster ovary cells.

It was possible that CT affected the Ca²⁺-dependent Cl channels directly. However, the decay time constant () of T_in measured before and after CT injection did not change (Fig. 6). Furthermore, oscillatory currents due to the Ca²⁺-activated Cl current were observed in oocytes after CT injection, consistent with lack of effect of CT on the Cl channel (46). Taken together, the possible involvement of the Ca²⁺-activated Cl channel was ruled out as a significant component of the inhibitory effect of CT on T_in.

Another explanation for the inhibition of Ca²⁺ release was that CT lowered cytosolic Ca²⁺ concentration to low levels by chelating Ca²⁺. Chelation of Ca²⁺ would prevent the Cl current evoked by Ca²⁺ released from its stores. However, Ca²⁺ chelation was unlikely to occur, because, in fact, an increase of [Ca²⁺]_i has been observed when CT was applied to Purkinje cells of rat cerebellum (48).

Ca²⁺ influx, enhanced by CT, could lead to levels of [Ca²⁺]_i high enough to block IP₃ receptors, because very high [Ca²⁺]_i has been reported to block IP₃ receptors completely (49, 50). If this mechanism occurred under our experimental conditions, however, the capacitative Ca²⁺ entry induced by 3-F-IP₃ or thapsigargin should have increased after CT injection. Such an increase was not observed (Figs. 2D and 5). Therefore, increased Ca²⁺ influx cannot account for the inhibitory effect of CT on IP₃-sensitive Ca²⁺ release. However, we cannot rule out the possibility that the Ca²⁺ influx enhanced by CT was large enough to block Ca²⁺ release through IP₃ receptor channels, but the change was too small to be detected by electrophysiological recording. This possibility remains to be investigated further.

We tested whether CT would affect directly Ca²⁺ release from IP₃-sensitive Ca²⁺ stores. The analysis shown in Fig. 7 revealed a strong, inverse relationship between time-to-peak of T_in measured before CT injection and residual current amplitude after CT injection. The response to IP₃ varied greatly from oocyte to oocyte, possibly due to the differences in the number and spatial distribution of IP₃ receptors (45, 51). Oocytes, which were highly sensitive to 3-F-IP_3, would deplete their Ca²⁺ stores more in response to a given concentration of 3-F-IP₃, compared with relatively insensitive oocytes. CT more potently blocked T_in (Ca²⁺ release from IP₃-sensitive stores) in oocytes displaying longer time-to-peak.

Two existing hypothesis can be adopted to explain the relationship between time-to-peak and sensitivity of each oocyte to IP₃, as follows: First, there is the steady-state model. It has been suggested that Ca²⁺ release is controlled by the intraluminal Ca²⁺level within Ca²⁺ stores (52). The steady-state model considered that decreasing the Ca²⁺ content of the stores would slow down further the Ca²⁺ release until it stopped when the level of luminal Ca²⁺ fell to a low concentration (53). Consequently, "sensitive" oocytes would have less full Ca²⁺ stores after injection of 3-F-IP₃ and would need more Ca²⁺ refilling to reach a certain threshold for Ca²⁺ release, which may result in delayed T_in (i.e. longer time-to-peak), as found (Fig. 7). Second, there is the feedback model. Feedback inhibition of the IP₃ receptor by cytosolic Ca²⁺ would limit further release of Ca²⁺, and recovery of sensitivity to IP₃ would follow the subsequent decline of cytosolic Ca²⁺ as the latter was re-sequestered (54, 55). Accordingly, oocytes highly sensitive to 3-F-IP₃ would have more Ca²⁺ liberated by injection of 3-F-IP₃. Such oocytes could take longer time to sequester Ca²⁺ to a non-blocking level of [Ca²⁺]_i and this may result in delayed T_in.

Thus, the time-to-peak can be considered as a measure of the IP₃ sensitivity of each oocyte. Ca²⁺ release in highly sensitive oocytes (where a large portion of Ca²⁺ store was exhausted by the 3-F-IP₃) was inhibited more by CT. Such a correlation would arise as a result of CT directly suppressing IP₃-sensitive Ca²⁺ release rather than one or more other auxiliary mechanisms. Hence, the inverse relationship found is consistent with CT inhibition being specific to the Ca²⁺ release mechanism from IP₃-sensitive Ca²⁺ stores. However, CT did not appear to inhibit directly the binding of 3-F-IP₃ to IP₃ receptors or compete with 3-F-IP₃ for IP₃ binding sites, because there was no significant effect of CT on I_t when co-injected with 3-F-IP₃, and high concentrations of 3-F-IP₃ could not overcome the CT-induced block. CT was most likely to uncouple the triggering action of Ca²⁺ influx from induction of the IP₃-sensitive Ca²⁺ release. It remains to be determined whether such an uncoupling effect occurs in vivo and whether it could lead to any long-term effect.

In overall conclusion, we have shown that the CT fragment of APP, but not the A fragments, suppresses Ca²⁺ release from IP₃-sensitive Ca²⁺ stores triggered by extracellular Ca²⁺ entry in the Xenopus oocyte model. Although the effect of CT peptide on Ca²⁺ release from IP₃-sensitive Ca²⁺ stores must be confirmed in the neuronal system, our results support further the view that the CT peptide plays an important role in pathogenesis of AD (56, 57). The inhibitory effect of CT on IP₃-sensitive Ca²⁺ release could disrupt intracellular Ca²⁺ homeostasis and thus contribute to the cellular toxicity in AD.

	FOOTNOTES

^* This work was supported in part by the National Creative Research Initiative Grant from the Ministry of Science & Technology, the BK21 Human Life Sciences Project, The British Council (Seoul), Korea, the Rotary Club International, and the Royal Society (to S. P. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 1051 Riverside Dr., New York, NY 10032.

To whom correspondence should be addressed. Tel.: 82-2-740-8285; Fax: 82-2-745-7996; E-mail: yhsuh@plaza.snu.ac.kr.

Published, JBC Papers in Press, April 1, 2002, DOI 10.1074/jbc.M108326200

	ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; IP₃, inositol 1,4,5-trisphosphate; 3-F-IP₃, D-3-deoxy-3-fluoro-myo-inositol 1,4, 5-trisphosphate; A, -amyloid protein; APP, amyloid precursor protein; CT, carboxyl-terminal; GST, glutathione S-transferase; TM, transmembrane.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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