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• W1968451839 abstract "In cytosol-like medium (CLM) with a free [Ca2+] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP3) (30 μm) evoked 45Ca2+ release from type 3 IP3 receptors only after a latency of 48 ± 6 ms; this latency could not be reduced by increasing the IP3concentration. In CLM containing a low free [Ca2+] (∼4 nm), 300 μm IP3 evoked45Ca2+ release after a latency of 66 ± 11 ms; this was reduced to 14 ± 3 ms when the [Ca2+] was 1 mm. Preincubation with CLM containing 100 μm Ca2+ caused a rapid (half-time = 33 ± 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP3 (300 μm). Hepatic (type 2) IP3 receptors were not inhibited by Ca2+ once they had bound IP3, but 100 μm Ca2+ rapidly inhibited type 3 IP3 receptors whether it was delivered before addition of IP3 or at any stage during a response to IP3. Ca2+ increases the affinity of IP3 for hepatic receptors by slowing IP3 dissociation, but Ca2+had no effect on IP3 binding to type 3 receptors. The rate of inhibition of type 3 IP3 receptors by Ca2+was faster than the rate of IP3 dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP3) agonist. Dissociation of agonist is not therefore required for Ca2+ to inhibit type 3 IP3 receptors. We conclude that type 2 and 3 IP3 receptors are each biphasically regulated by Ca2+, but by different mechanisms. For both, IP3 binding causes a stimulatory Ca2+-binding site to be exposed allowing Ca2+ to bind and open the channel. IP3 binding protects type 2 receptors from Ca2+ inhibition, but type 3 receptors are inhibited by Ca2+ whether or not they have IP3 bound. Increases in cytosolic [Ca2+] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP3 has dissociated. In cytosol-like medium (CLM) with a free [Ca2+] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP3) (30 μm) evoked 45Ca2+ release from type 3 IP3 receptors only after a latency of 48 ± 6 ms; this latency could not be reduced by increasing the IP3concentration. In CLM containing a low free [Ca2+] (∼4 nm), 300 μm IP3 evoked45Ca2+ release after a latency of 66 ± 11 ms; this was reduced to 14 ± 3 ms when the [Ca2+] was 1 mm. Preincubation with CLM containing 100 μm Ca2+ caused a rapid (half-time = 33 ± 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP3 (300 μm). Hepatic (type 2) IP3 receptors were not inhibited by Ca2+ once they had bound IP3, but 100 μm Ca2+ rapidly inhibited type 3 IP3 receptors whether it was delivered before addition of IP3 or at any stage during a response to IP3. Ca2+ increases the affinity of IP3 for hepatic receptors by slowing IP3 dissociation, but Ca2+had no effect on IP3 binding to type 3 receptors. The rate of inhibition of type 3 IP3 receptors by Ca2+was faster than the rate of IP3 dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP3) agonist. Dissociation of agonist is not therefore required for Ca2+ to inhibit type 3 IP3 receptors. We conclude that type 2 and 3 IP3 receptors are each biphasically regulated by Ca2+, but by different mechanisms. For both, IP3 binding causes a stimulatory Ca2+-binding site to be exposed allowing Ca2+ to bind and open the channel. IP3 binding protects type 2 receptors from Ca2+ inhibition, but type 3 receptors are inhibited by Ca2+ whether or not they have IP3 bound. Increases in cytosolic [Ca2+] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP3 has dissociated. Inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3inositol 1,4,5-trisphosphateCLMcytosol-like mediumEC50half-maximally effective concentrationPipespiperazine-N,N′-bis(2-ethanesulfonic acid) receptors belong to a family of intracellular channels that mediate the release of Ca2+ from intracellular stores in response to a range of physiological stimuli (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). The three mammalian subtypes of the IP3 receptor (types 1–3), which form both homotetrameric (2Hirota J. Michikawa T. Miyawaki A. Furuichi T. Okura I. Mikoshiba K. J. Biol. Chem. 1995; 270: 19046-19051Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and heterotetrameric (3Monkawa T. Miyawaki A. Sugiyama T. Yoneshima H. Yamamoto-Hino M. Furuichi T. Saruta T. Hasagawa M. Mikoshiba K. J. Biol. Chem. 1995; 270: 14700-14704Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 4Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res. Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar) complexes, are regulated by both IP3 and Ca2+ (5Taylor C.W. Biochim. Biophys. Acta. 1998; 1436: 19-33Crossref PubMed Scopus (146) Google Scholar). This interplay between IP3 and Ca2+ is likely to determine the complex Ca2+ signals evoked by receptors that stimulate IP3 formation. inositol 1,4,5-trisphosphate cytosol-like medium half-maximally effective concentration piperazine-N,N′-bis(2-ethanesulfonic acid) The similarities between the IP3 receptor subtypes are presently more striking than the differences. Each subtype is predicted to form a similar structure with an N-terminal IP3-binding domain separated by some 1500 residues from a C-terminal region that includes six membrane-spanning domains, the last two of which together with the intervening loop line the pore of the channel (6Galvan D.L. Borrego-Diaz E. Perez P.J. Mignery G.A. J. Biol. Chem. 1999; 274: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 7Boehning D. Mak D.-O. D. Foskett J.K. Joseph S.K. J. Biol. Chem. 2001; 276: 13509-13512Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Within the functional receptor, however, the IP3-binding domain of one subunit appears to be intimately associated with the pore region of a neighboring subunit (8Boehning D. Joseph S.K. EMBO J. 2000; 19: 5450-5459Crossref PubMed Scopus (99) Google Scholar). The ion permeation properties of the channel are also similar for each receptor subtype (9Ramos-Franco J. Fill M. Mignery G.A. Biophys. J. 1998; 75: 834-839Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 10Mak D.-O., D. McBride S. Raghiram V. Yue Y. Joseph S.K. Foskett J.K. J. Gen. Physiol. 2000; 115: 241-255Crossref PubMed Scopus (74) Google Scholar), consistent with the highly conserved sequences found within the pore-forming region (11Taylor C.W. Genazzani A.A. Morris S.A. Cell Calcium. 1999; 26: 237-251Crossref PubMed Scopus (245) Google Scholar). The most important differences between IP3 receptor subtypes, which are certainly differentially expressed (11Taylor C.W. Genazzani A.A. Morris S.A. Cell Calcium. 1999; 26: 237-251Crossref PubMed Scopus (245) Google Scholar), are therefore likely to be in their modulation (12Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (373) Google Scholar, 13Cardy T.J.A. Taylor C.W. Biochem. J. 1998; 334: 447-455Crossref PubMed Scopus (66) Google Scholar, 14Yule D.I. J. Gen. Physiol. 2001; 117: 431-434Crossref PubMed Scopus (27) Google Scholar, 15Mak D.-O. McBride S. Foskett J.K. J. Gen. Physiol. 2001; 117: 435-446Crossref PubMed Scopus (117) Google Scholar) and perhaps in their subcellular distribution (16Hirata K. Nathanson M.H. Burgstahler A.D. Okazaki K. Mattei E. Sears M.L. Invest. Opthalmol. Vis. Sci. 1999; 40: 2046-2053PubMed Google Scholar, 17El-Daher S.S. Patel Y. Siddiqua A. Hassock S. Edmunds S. Maddison B. Patel G. Goulding D. Lupu F. Wojcikiewicz J.H. Authi K.S. Blood. 2000; 95: 3412-3422Crossref PubMed Google Scholar). Biphasic regulation of IP3 receptors by cytosolic Ca2+ is widespread (5Taylor C.W. Biochim. Biophys. Acta. 1998; 1436: 19-33Crossref PubMed Scopus (146) Google Scholar), with many reports confirming that modest increases in Ca2+ stimulate channel opening, while more substantial increases are inhibitory for type 1 (18Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1441) Google Scholar) and type 2 (19Marshall I.C.B. Taylor C.W. J. Biol. Chem. 1993; 268: 13214-13220Abstract Full Text PDF PubMed Google Scholar) IP3 receptors, as well as for the IP3receptors from Xenopus (20Mak D.-O., D. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (230) Google Scholar) and insects (21Swatton J.E. Morris S.A. Taylor C.W. Biochem. J. 2001; 359: 435-441Crossref PubMed Scopus (20) Google Scholar, 22Baumann O. Walz B. J. Comp. Physiol. A. 1989; 165: 627-636Crossref Scopus (60) Google Scholar). The effects of Ca2+ on type 3 IP3 receptors have aroused more controversy. In bilayer recordings from the type 3 IP3 receptors of RIN-5F cells, even very high concentrations of Ca2+ failed to inhibit channel activity (23Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 296: 81-84Crossref Scopus (230) Google Scholar). IP3-evoked Ca2+ release from DT40 cells lacking types 1 and 2 IP3 receptors also appeared to be resistant to Ca2+ inhibition (24Miyakawa T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Iino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (342) Google Scholar). But many other studies of both cells expressing predominantly type 3 IP3 receptors (25Missiaen L. Parys J.B. Sienaert I. Maes K. Kunzelmann K. Takahashi M. Tanzawa K. De Smet H. J. Biol. Chem. 1998; 273: 8983-8986Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), including RINm5F cells (26Swatton J.E. Morris S.A. Cardy T.J.A. Taylor C.W. Biochem. J. 1999; 344: 55-60Crossref PubMed Scopus (40) Google Scholar), and of recombinant type 3 IP3 receptors (15Mak D.-O. McBride S. Foskett J.K. J. Gen. Physiol. 2001; 117: 435-446Crossref PubMed Scopus (117) Google Scholar, 27Boehning D. Joseph S.K. J. Biol. Chem. 2000; 275: 21492-21499Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) have reported biphasic regulation of IP3 receptor behavior by cytosolic Ca2+. It seems likely, therefore, that biphasic regulation by cytosolic Ca2+ may be an ubiquitous feature of IP3 receptors, although it is far from clear that the underlying mechanisms are the same (28Yoneshima H. Miyawaki A. Michikawa T. Furuichi T. Mikoshiba K. Biochem. J. 1997; 322: 591-596Crossref PubMed Scopus (88) Google Scholar, 29Cardy T.J.A. Traynor D. Taylor C.W. Biochem. J. 1997; 328: 785-793Crossref PubMed Scopus (99) Google Scholar). The rapid kinetics of IP3 receptor regulation by IP3 and Ca2+ are likely to be significant in determining the contributions of different IP3 receptor subtypes to the complex regenerative Ca2+ signals in intact cells (30Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar, 31Adkins C.E. Taylor C.W. Curr. Biol. 1999; 9: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Despite the importance of resolving the behavior of IP3 receptors on a time scale appropriate to understanding rapid Ca2+ release events in intact cells (30Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar), most studies of IP3 receptor gating have been limited to either steady-state measurements (15Mak D.-O. McBride S. Foskett J.K. J. Gen. Physiol. 2001; 117: 435-446Crossref PubMed Scopus (117) Google Scholar, 23Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 296: 81-84Crossref Scopus (230) Google Scholar) or to examining rates of Ca2+ release with very limited temporal resolution. We previously used rapid superfusion of permeabilized rat hepatocytes loaded with 45Ca2+ to examine the kinetics of IP3-evoked Ca2+ release by type 2 IP3 receptors with a temporal resolution of 9 ms (31Adkins C.E. Taylor C.W. Curr. Biol. 1999; 9: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 32Marchant J.S. Taylor C.W. Curr. Biol. 1997; 7: 510-518Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We concluded that IP3 binding determined whether Ca2+ stimulated or inhibited channel opening (31Adkins C.E. Taylor C.W. Curr. Biol. 1999; 9: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 32Marchant J.S. Taylor C.W. Curr. Biol. 1997; 7: 510-518Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Here, we apply similar methods to examine IP3-evoked Ca2+ release from permeabilized RINm5F cells, which both we (26Swatton J.E. Morris S.A. Cardy T.J.A. Taylor C.W. Biochem. J. 1999; 344: 55-60Crossref PubMed Scopus (40) Google Scholar) and others (33Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) have shown to express predominantly type 3 IP3 receptors. IP3 was from American Radiolabeled Chemicals (St. Louis, MO). 45Ca2+(238.5 Ci/mol) was from ICN (Thame, Oxfordshire, UK) and [3H]IP3 (37 Ci/mmol) was from Amersham Pharmacia Biotech (Little Chalfont, UK). Ionomycin, synthetic adenophostin A, and 3-deoxy-3-fluoro IP3 were from Calbiochem (Nottingham, UK). Thapsigargin was from Alomone Labs (Jerusalem, Israel). All other materials were from suppliers listed earlier (26Swatton J.E. Morris S.A. Cardy T.J.A. Taylor C.W. Biochem. J. 1999; 344: 55-60Crossref PubMed Scopus (40) Google Scholar). RINm5F rat insulinoma cells (a gift from Dr. Peter Brown, University of Manchester, UK) were cultured at 37 °C in 5% CO2 in RPMI 1640 medium containing 2 mml-glutamine (Invitrogen, Paisley, UK) and 5% fetal calf serum (Sigma, Poole, UK). Cells were passaged every 3–4 days when confluent. Hepatocytes were isolated from the livers of male Wistar rates (200–300 g) by collagenase digestion (34Taylor C.W. Marchant J.S. Milligan G. Signal Transduction: A Practical Approach. IRL Press, Oxford1999: 361-384Google Scholar) and kept for up to 6 h at 4 °C in Eagle's minimal essential medium containing 26 mm NaHCO3 and 2% bovine serum albumin. Confluent RINm5F cells (passages 90–100) were scraped from a flask, washed by centrifugation (650 × g, 2 min), and resuspended (1 × 106 cells/ml) in Ca2+-free cytosol-like medium (CLM: 140 mm KCl, 20 mm NaCl, 2 mmMgCl2, 1 mm EGTA, 20 mm Pipes, pH 7.0, at 37 °C). The cells were permeabilized by incubation with saponin (10 μg/ml) for 5 min at 37 °C (26Swatton J.E. Morris S.A. Cardy T.J.A. Taylor C.W. Biochem. J. 1999; 344: 55-60Crossref PubMed Scopus (40) Google Scholar), washed by centrifugation (650 × g, 2 min), and resuspended in CLM containing ∼200 nm free Ca2+ (300 μm total Ca2+), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (10 μm), and 45Ca2+ (10 μCi/ml). After addition of ATP (1.5 mm), creatine phosphate (5 mm), and creatine phosphokinase (5 units/ml), cells were incubated for 15 min at 37 °C during which their intracellular stores reached a steady-state Ca2+ content of 141 ± 14 pmol/106 cells (n = 3). Cells were rapidly removed from the loading medium by 8-fold dilution into CLM (200 nm free Ca2+, 20 °C) and a brief centrifugation (650 × g, 30 s), before resuspension (6 × 106 cells/ml) in CLM (200 nm free Ca2+, 20 °C). The effects of IP3 or its analogues on the 45Ca2+content of the stores were determined after 2-min incubations at 20 °C (to allow direct comparison with superfusion experiments) and in the presence of thapsigargin (1 μm) to inhibit the endoplasmic reticulum Ca2+-ATPase. The incubations were terminated by rapid filtration through Whatman GF/C filters using a Brandel receptor binding harvester (26Swatton J.E. Morris S.A. Cardy T.J.A. Taylor C.W. Biochem. J. 1999; 344: 55-60Crossref PubMed Scopus (40) Google Scholar), and the45Ca2+ content of the stores determined by liquid scintillation counting. Active 45Ca2+accumulation was defined as the 45Ca2+ that could be released by ionomycin (10 μm). Permeabilized RINm5F cells loaded with45Ca2+ (30 μCi/ml) were immobilized on a nitrocellulose and glass fiber filter array held in the chamber of a rapid superfusion apparatus; the apparatus has been described elsewhere (34Taylor C.W. Marchant J.S. Milligan G. Signal Transduction: A Practical Approach. IRL Press, Oxford1999: 361-384Google Scholar). Briefly it allows rates of 45Ca2+ release from the immobilized cells to be measured with a temporal resolution of up to 9 ms, as CLM flows (at 2 ml/s) from pressurized cylinders to the cells and then (with the 45Ca2+ released from the cells) into vials arranged around a circular fraction collector. Because the medium bathing the immobilized cells is continuously replaced during superfusion, unidirectional45Ca2+ efflux can be measured without the addition of thapsigargin used in the conventional45Ca2+ efflux experiments (see above). Inclusion of an inert marker ([3H]inulin) in the superfusing media allowed the arrival of a stimulus to be precisely related to changes in 45Ca2+ efflux. Under the conditions used for these experiments, the half-time (t1/2) for exchange of the media was 31 ± 1 ms (n = 3). All experiments were performed at 20 °C. The size of the intracellular 45Ca2+ pool was calculated for each experiment by summing all45Ca2+ released during the stimulation with that released at the end of the experiment by Triton X-100 (0.5%). In some experiments (Fig. 2) very rapid increases in free [Ca2+] were achieved using a pulsing protocol (31Adkins C.E. Taylor C.W. Curr. Biol. 1999; 9: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar): a 50-ms pulse of CLM containing 1.65 mm Ca2+followed by continuous superfusion with CLM containing 1.1 mm Ca2+ allowed the free [Ca2+] to be increased from ∼200 nm to 100 μmwithin 50 ms. Permeabilized hepatocytes were prepared and loaded with45Ca2+ as reported previously (34Taylor C.W. Marchant J.S. Milligan G. Signal Transduction: A Practical Approach. IRL Press, Oxford1999: 361-384Google Scholar), and rates of IP3-evoked 45Ca2+ release were then measured using rapid superfusion as described for RINm5F cells. Free [Ca2+] were predicted using the computer program WinMAXC version 2.05 (C. Patton, Stanford University, CA 93950), and then measured using either fura 2 or a Ca2+-sensitive electrode (19Marshall I.C.B. Taylor C.W. J. Biol. Chem. 1993; 268: 13214-13220Abstract Full Text PDF PubMed Google Scholar, 26Swatton J.E. Morris S.A. Cardy T.J.A. Taylor C.W. Biochem. J. 1999; 344: 55-60Crossref PubMed Scopus (40) Google Scholar). Saponin-permeabilized RINm5F cells were washed by centrifugation (650 × g, 2 min), resuspended in TE medium (50 mm Tris, 1 mm EDTA, pH 8.3, at 2 °C), and incubated (2.75 × 106 cells in 200 μl) with [3H]IP3 (3 nm) and competing ligands for 5 min at 2 °C. Bound and free ligand were separated by filtration (35Nerou E.P. Riley A.M. Potter B.V.L. Taylor C.W. Biochem. J. 2001; 355: 59-69Crossref PubMed Scopus (37) Google Scholar), and 3H activity was determined by liquid scintillation counting. Specific [3H]IP3 binding (∼500 disintegrations/min) was typically ∼70% of total binding. Equilibrium binding data were analyzed to provide dissociation constants (Kd) for competing ligands as reported previously (35Nerou E.P. Riley A.M. Potter B.V.L. Taylor C.W. Biochem. J. 2001; 355: 59-69Crossref PubMed Scopus (37) Google Scholar). Because the 45Ca2+ content of the stores declines as a superfusion experiment progresses, we express many of the responses to IP3 as fractional release rates, where the amount of 45Ca2+ released by IP3 is expressed as a fraction of the45Ca2+ remaining within the IP3-sensitive stores at the beginning of that interval (34Taylor C.W. Marchant J.S. Milligan G. Signal Transduction: A Practical Approach. IRL Press, Oxford1999: 361-384Google Scholar). By expressing rates of 45Ca2+ release relative to the amount of 45Ca2+ available for release, this form of analysis effectively isolates the activity of the IP3 receptor from changes in the45Ca2+ content of the stores. We thereby expect stable fractional release rates unless the IP3 receptor changes its behavior (36Marchant J.S. Taylor C.W. Biochemistry. 1998; 37: 11524-11533Crossref PubMed Scopus (56) Google Scholar). IP3-evoked Ca2+ release was calculated by subtracting the basal rate of 45Ca2+ release from the 45Ca2+ detected in each fraction during stimulation with IP3. By measuring basal rates of45Ca2+ release over protracted times (not shown), we established the following empirical relationship between the fraction of the Ca2+ stores released (S), the fractional rate of unstimulated 45Ca2+ release at the beginning of the superfusion (I) and the fractional rate of unstimulated 45Ca2+ release from partially depleted stores (P). P=I (0.71e−8.8S+0.31)Equation 1 This equation was used to separately compute the basal rate of45Ca2+ release from IP3-sensitive and IP3-insensitive Ca2+ stores throughout the experiment. Concentration-effect relationships were fitted to logistic equations using nonlinear curve fitting (KaleidaGraph, Synergy Software, PA) (37Marchant J.S. Beecroft M.D. Riley A.M. Jenkins D.J. Marwood R.D. Taylor C.W. Potter B.V.L. Biochemistry. 1997; 36: 12780-12790Crossref PubMed Scopus (67) Google Scholar). In permeabilized RINm5F cells, IP3 caused a rapid increase in the rate of 45Ca2+ release, with the half-maximal rate occurring with an IP3 concentration of 4.96 ± 0.82 μm (n = 3) and the maximal rate of 45Ca2+ release occurring with 10 μm IP3. During stimulation with 30 μm IP3 in normal CLM, the cells were therefore exposed to a supramaximal concentration of IP3within about 15 ms, but the first detectable release of45Ca2+ occurred only after a delay of 48 ± 6 ms (n = 3) (Fig. 1A, i). This long absolute latency, which is similar to that observed previously for hepatic IP3 receptors (32Marchant J.S. Taylor C.W. Curr. Biol. 1997; 7: 510-518Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), was not shortened by further increasing the IP3 concentration (Fig. 1A, ii): the latency was 43 ± 9 ms (n = 3) when cells were stimulated with 300 μm IP3 (Table I). In the latter experiments, cells would have been exposed to at least 7 times the maximally effective IP3 concentration (∼70 μm) within 10 ms.Table ICytosolic Ca2+ shortens the latency for IP3 receptor activation without affecting the peak rate of 45Ca2+releaseFree [Ca2+]30 μm IP3, Latency300 μm IP3LatencyTime to peakPeak ratemsmsms%/10 ms∼4 nm66 ± 1266 ± 11217 ± 70.35 ± 0.09200 nm48 ± 643 ± 9293 ± 30.50 ± 0.04100 μmND1-aND, not determined.35 ± 4170 ± 150.44 ± 0.071 mmND14 ± 3121 ± 130.46 ± 0.08Latencies, defined as the first of 3 successive rises in45Ca2+ release following detection of [3H]inulin (included with the IP3) in the superfusate, are shown for cells stimulated with 30 or 300 μm IP3. Where indicated, the free [Ca2+] was increased (from 200 nm to 100 μm or from nominally Ca2+-free to 1 mm) by simultaneous delivery of IP3 and CLM containing high-Ca2+. For cells stimulated in CLM containing ∼4 or 200 nm free Ca2+, cells were preincubated for at least 10 s with the appropriate CLM before delivery of IP3 in the same medium. Peak rates of 45Ca2+release evoked by 300 μm IP3 and the time to reach the peak rate are also shown. Mean ± S.E. of three to six independent determinations.1-a ND, not determined. Open table in a new tab Latencies, defined as the first of 3 successive rises in45Ca2+ release following detection of [3H]inulin (included with the IP3) in the superfusate, are shown for cells stimulated with 30 or 300 μm IP3. Where indicated, the free [Ca2+] was increased (from 200 nm to 100 μm or from nominally Ca2+-free to 1 mm) by simultaneous delivery of IP3 and CLM containing high-Ca2+. For cells stimulated in CLM containing ∼4 or 200 nm free Ca2+, cells were preincubated for at least 10 s with the appropriate CLM before delivery of IP3 in the same medium. Peak rates of 45Ca2+release evoked by 300 μm IP3 and the time to reach the peak rate are also shown. Mean ± S.E. of three to six independent determinations. While the latency could not be reduced by increasing the IP3 concentration, it was reduced and ultimately abolished by increasing the free [Ca2+] of the CLM. In nominally Ca2+-free CLM (free [Ca2+] ∼4 nm), the latency after stimulation with 300 μm IP3 was 66 ± 11 ms and this was reduced as the free [Ca2+] was increased (Table I), such that when the free [Ca2+] was 1 mm, the latency was only 14 ± 3 ms (Table I, Fig. 1B). It is noteworthy that although increasing the free [Ca2+] shortened both the latency and the time taken for the rate of45Ca2+ release to reach its peak, it had no significant effect on the peak rate of 45Ca2+release (Table I). Although there was an immediate rise in the rate of45Ca2+ release when IP3 (300 μm) was delivered in the presence of 1 mmfree Ca2+, there was a lag of ∼40 ms before the response attained its fastest rate (Fig. 1B). We considered whether this slow take-off might simply result from a relatively slow increase in free [Ca2+] as Ca2+-free CLM (1 mm EGTA) was replaced by (t1/2 = 31 ± 1 ms) the high-Ca2+ CLM (2 mmCa2+, 1 mm EGTA) causing an abrupt increase in free [Ca2+] as the buffering capacity of the EGTA was exceeded. The simulation shown in Fig. 1D confirms that under the conditions used for these experiments, a switch to high-Ca2+ CLM caused the free [Ca2+] surrounding the cells to abruptly increase to several micromolar only after a delay of ∼25 ms. By reducing the EGTA concentration in the CLM used initially to 100 μmand then stepping to CLM containing 1 mm Ca2+without EGTA, the free [Ca2+] surrounding the cells was predicted to increase almost linearly to 1 mm and to exceed 100 μm within 8 ms (Fig. 1E). We used this Ca2+-delivery protocol to very rapidly expose cells to a high free [Ca2+] during the latent period of the response to a supramaximal concentration of IP3. Cells were first exposed to IP3 (300 μm) in nominally Ca2+-free CLM (100 μm EGTA) for 30 ms and then to high-Ca2+ in EGTA-free CLM (Fig. 1C). During the initial exposure to IP3, there was no stimulated release of 45Ca2+ because this interval lies within the latent period (∼66 ms in nominally Ca2+-free CLM). After rapidly increasing the free [Ca2+], there was an immediate increase in the rate of45Ca2+ release with no detectable latency (Fig. 1C). Stimulation of permeabilized RINm5F cells with a maximal concentration of IP3 (10 μm) caused a rapid increase in the rate of 45Ca2+ release to a peak, followed by a decay over several seconds (Fig. 2A). Preincubation of the cells for 1.2 s with CLM containing a free [Ca2+] of 100 μm completely abolished the subsequent response to IP3 (Fig. 2A). The increase in free [Ca2+] itself only minimally increased the rate of45Ca2+ release from 0.16 ± 0.01%/100 ms to 0.23 ± 0.01%/100 ms (n = 15) (Fig. 2D), indicating that depletion of intracellular Ca2+ stores could not account for the loss of response to IP3, and (see below) that increasing the free [Ca2+] does not itself significantly stimulate45Ca2+ release in the absence of IP3. The reversibility of the inhibition under conditions where the stores could not re-load with Ca2+ (see below) further confirms that the lack of response to IP3 results from inhibition of IP3 receptors rather than store depletion. The inhibition of IP3 receptors by high Ca2+could not be overcome by increasing the concentration of IP3 (Fig. 2B). The normal peak rate of45Ca2+ release evoked by 10 μmIP3 was 3.03 ± 0.15%/100 ms (n = 3). After preincubation with high Ca2+ for 1.2 s, the peak rate of Ca2+ release fell to 0.027 ± 0.008%/100 ms (n = 3) after stimulation with 10 μmIP3, and to 0.24 ± 0.07%/100 ms (n = 3) after stimulation with 300 μm IP3. Inhibition of IP3 receptors by Ca2+ has been proposed to be mediated by calmodulin (38Missiaen L. Parys J.B. Weidema A.F. Sipma H. Vanlingen S., De Smet P. Callewaert G. De Smedt H. J. Biol. Chem. 1999; 274: 13748-13751Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 39Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), by other Ca2+-binding proteins (40Danoff S.K. Supattapone S. Snyder S.H. Biochem. J. 1988; 254: 701-705Crossref PubMed Scopus (118) Google Scholar) or by residues within the IP3 receptor (41Miyakawa T. Mizushima A. Hirose K. Yamazawa T. Bezprozvanny I. Kurosaki T. Iino M. EMBO J. 2001; 20: 1674-1680Crossref PubMed Scopus (118) Google Scholar). We have not established the site through which Ca2+ inhibits type 3 IP3 receptors, although it is unlikely to be calmodulin because neither calmidazolium (20 μm) nor a peptide inhibitor derived from Ca2+-calmodulin-dependent protein kinase (10 μm) (13Cardy T.J.A. Taylor C.W. Biochem. J. 1998; 334: 447-455Crossref PubMed Scopus (66) Google Scholar) prevented complete inhibition of the IP3 receptor by CLM containing 100 μmCa2+ (not shown). By varying the duration of the preincubation with high-Ca2+ CLM before stimulating with 10 μmIP3, the half-time for inhibition by cytosolic Ca2+ was established (39 ± 3 ms, n = 3) (Fig. 2C). We were concerned that with such rapid inhibition, the time taken for IP3 to reach its maximally effective concentration was likely to significantly affect our measurement of the kinetics of Ca2+ inhibition. To resolve the issue, the time course of Ca2+ inhibition was investigated by preincubating with high-Ca2+ CLM and then assessing IP3 receptor activity using 300 μmIP3, which allowed the IP3 concentration to exceed 70 μm within 10 ms. The half-time for Ca2+ inhibition determined using this method (33 ± 9 ms) was indistinguishable from that observed using 10 μmIP3 as the test pulse (39 ± 3 ms). To a" @default.
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• W1968451839 title "Fast Biphasic Regulation of Type 3 Inositol Trisphosphate Receptors by Cytosolic Calcium" @default.
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