Ca2+ signalling between single L-type Ca2+ channels and ryanodine ...

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Received 11 December 2000; accepted 6 February 2001. 1. Matsuda, L. A., Lolait, S. J., Brownstein, M. J., Young, A. C. & Bonner, T. I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561±564 (1990). 2. Caul®eld, M. P. & Brown, D. A. Cannabinoid receptor agonists inhibit Ca current in NG108-15 neuroblastoma cells via a pertussis toxin-sensitive mechanism. Br. J. Pharmacol. 106, 231±232 (1992). 3. Mackie, K. & Hille, B. Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells. Proc. Natl Acad. Sci. USA 89, 3825±3829 (1992). 4. Herkenham, M. et al. Cannabinoid receptor localization in brain. Proc. Natl Acad. Sci. USA 87, 1932± 1936 (1990). 5. Devane, W. A. et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946±1949 (1992). 6. Stella, N., Schweitzer, P. & Piomelli, D. A second endogenous cannabinoid that modulates long-term potentiation. Nature 388, 773±778 (1997). 7. Di Marzo, V. et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686±691 (1994). 8. Di Marzo, V., Melck, D., Bisogno, T. & De Petrocellis, L. Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action. Trends Neurosci. 21, 521±528 (1998). 9. Tsou, K., Mackie, K., SanÄudo-PenÄa, M. C. & Walker, J. M. Cannabinoid CB1 receptors are localized primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation. Neuroscience 93, 969±975 (1999). 10. Katona, I. et al. Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of speci®c hippocampal interneurons. J. Neurosci. 19, 4544±4558 (1999). 11. Hajos, N. et al. Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations. Eur. J. Neurosci. 12, 3239±3249 (2000). 12. Hoffman, A. F. & Lupica, C. R. Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. J. Neurosci. 20, 2470±2479 (2000). 13. Pitler, T. A. & Alger, B. E. Postsynaptic spike ®ring reduces synaptic GABAA responses in hippocampal pyramidal cells. J. Neurosci. 12, 4122±4132 (1992). 14. Lenz, R. A., Wagner, J. J. & Alger, B. E. N- and L-type calcium channel involvement in depolarization-induced suppression of inhibition in rat hippocampal CA1 cells. J. Physiol. 512, 61±73 (1998). 15. Alger, B. E. et al. Retrograde signalling in depolarization-induced suppression of inhibition in rat hippocampal CA1 cells. J. Physiol. 496, 197±209 (1996). 16. Morishita, W. & Alger, B. E. Sr2+ supports depolarization-induced suppression of inhibition and provides new evidence for a presynaptic expression mechanism in rat hippocampal slices. J. Physiol. 505, 307±317 (1997). 17. Pitler, T. A. & Alger, B. E. Depolarization-induced suppression of GABAergic inhibition in rat hippocampal pyramidal cells: G protein involvement in a presynaptic mechanism. Neuron 13, 1447± 1455 (1994). 18. Beltramo, M. et al. Functional role of high-af®nity anandamide transport, as revealed by selective inhibition. Science 277, 1094±1097 (1997). 19. Piomelli, D. et al. Structural determinants for recognition and translocation by the anandamide transporter. Proc. Natl Acad. Sci. USA 96, 5802±5807 (1999). 20. Binz, T. et al. Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J. Biol. Chem. 269, 1617± 1620 (1994). 21. Leung, S. M., Chen, D., DasGupta, B. R., Whiteheart, S. W. & Apodaca, G. SNAP-23 requirement for transferrin recycling in Streptolysin-O-permeabilized Madin-Darby canine kidney cells. J. Biol. Chem. 273, 17732±17741 (1998). 22. Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759±772 (1998). 23. Lledo, P. M., Zhang, X., SuÈdhof, T. C., Malenka, R. C. & Nicoll, R. A. Postsynaptic membrane fusion and long-term potentiation. Science 279, 399±403 (1998). 24. LuÈscher, C. et al. Role of AMPA receptor cycling in synaptic transmission and plasticity. Neuron 24, 649±658 (1999). 25. Morishita, W., Kirov, S. A. & Alger, B. E. Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1. J. Neurosci. 18, 4870±4882 (1998). 26. Morishita, W. & Alger, B. E. Differential effects of the group II mGluR agonist, DCG-IV, on depolarization-induced suppression of inhibition in hippocampal CA1 and CA3 neurons. Hippocampus 10, 261±268 (2000). 27. Vincent, P. & Marty, A. Neighboring cerebellar Purkinje cells communicate via retrograde inhibition of common presynaptic interneurons. Neuron 11, 885±893 (1993). 28. Cash, S., Zucker, R. S. & Poo, M. M. Spread of synaptic depression mediated by presynaptic cytoplasmic signaling. Science 272, 998±1001 (1996). 29. WigstroÈm, H. & Gustafsson, B. Facilitation of hippocampal long-lasting potentiation by GABA antagonists. Acta Physiol. Scand. 125, 159±172 (1985). 30. Brody, D. L. & Yue, D. T. Relief of G-protein inhibition of calcium channels and short-term synaptic facilitation in cultured hippocampal neurons. J. Neurosci. 20, 889±898 (2000).

Acknowledgements We thank J. S. Isaacson and E. Schnell for suggesting a role for cannabinoids in DSI; R. S. Zucker for technical advice in the calcium uncaging experiments; and R. H. Scheller and Y. A. Chen for the gift of recombinant BTE light chain and SNAP-25. We are grateful for the comments on the manuscript contributed by D. S. Bredt, D. R. Copenhagen, R. H. Edwards, M. Frerking, D. Schmitz and M. P. Stryker. R.I.W. is supported by a National Science Foundation Graduate Research Fellowship. R.A.N. is a member of the Keck Center for Integrative Neuroscience and the Silvio Conte Center for Neuroscience Research. R.A.N. was supported by grants from the National Institutes of Health and the Bristol-Myers Squibb Corporation. Correspondence and requests for materials should be addressed to R.A.N. (e-mail: [email protected]).

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Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells Shi-Qiang Wang*, Long-Sheng Song*, Edward G. Lakatta* & Heping Cheng*² * Laboratory of Cardiovascular Sciences, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA ² National Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China ..............................................................................................................................................

Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals1±5. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum3±5. The Ca2+ in¯ux through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks6. Here we determine the kinetics, ®delity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a `Ca2+ sparklet', can trigger about 4±6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of singlechannel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level. To visualize the trigger Ca2+ entry through single LCCs in intact rat ventricular myocytes, we used confocal imaging in the line-scan mode to detect the local Ca2+ signal directly beneath the voltageclamped patch membrane7 (Fig. 1, inset). The sarcoplasmic reticulum (SR) Ca2+ release was paralysed by 10 mM caffeine and 10 mM thapsigargin, an SR Ca2+pump blocker. When the pipette solution contained 20 mM Ca2+ and 10 mM FPL64176 (FPL), an LCC agonist that prolongs channel open time8,9, patch depolarization from a holding potential of -50 mV to test potentials between -40 and 0 mV activated single-channel currents (iCa) in a voltage-dependent manner (Figs 1 and 2a). Each open event was accompanied by a discrete minuscule Ca2+ transient, which we named a `Ca2+ sparklet', in close proximity to the patch membrane. Ca2+ sparklets were resistant to ryanodine (10 mM, 5 min, n = 3 patches), but were completely abolished by the LCC antagonist, nifedipine (2 mM, 3 min, n = 3 patches). These data indicate that Ca2+ sparklets originate from single LCC openings, and represent the ®rst optical measurement of voltage-gated single-channel activity in intact cells. Unlike Ca2+ sparks of ryanodine receptor (RyR) origin, which typically last for ,30 ms (ref. 6), Ca2+ sparklets have a variable duration: the onset and termination of Ca2+ sparklets correlated tightly with the open and closure of the channel, respectively (Fig. 1). To characterize sparklet properties in relation to Ca2+ in¯ux, we measured the `signal mass' of sparklets (that is, the space-time integral of local DF/F0 of a sparklet in the line-scan image), and integrated the corresponding unitary Ca2+ in¯ux (qCa). Over the voltage range from -30 mV to +10 mV, at which LCC openings can be clearly identi®ed, there was a linear correlation between qCa (,8,000±100,000 Ca2+ ions) and sparklet signal mass (Fig. 2b). Hence, Ca2+ sparklets provide a faithful readout of LCC unitary current. In heart cells, Ca2+ sparklets from single LCCs are expected to deliver trigger signal into the 12-nm junctional cleft10,11 to activate

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sparks of RyR origin. The simultaneous visualization of sparklets and triggered sparks provides a unique opportunity to examine the control of SR Ca2+ release at the molecular level. Either inhibition of LCC in¯ux by 2 mM nifedipine (Fig. 4b) or substitution of the pipette Ca2+ with 20 mM Ba2+ (Fig. 4c) extinguished spark activation, indicating that Ca2+ sparks are triggered by Ca2+ entry through LCCs, but not by voltage per se. To de®ne the exact temporal relationship between the sparklet and the triggered spark, we obtained confocal images at an improved time resolution (0.7 ms per line) and a high signal-tonoise ratio (Fig. 5). Figure 5 shows that a slow-rising `foot', which was not seen in spontaneous sparks, preceded the rapid upstroke of most triggered sparks. The similarity between the initial rate of rise of the feet and that of solitary sparklets (Fig. 6a, c) con®rmed that the foot is a sparklet that triggers a spark. The latency (L) from the onset of a sparklet foot to its triggered Ca2+ spark was well described by a single-exponential function, with a time constant t = 6.7 ms (Fig. 6b). The exponentially ®tted curve in Fig. 6b fully accounts for the triggered sparks with a sparklet foot # 2.8 ms (counted in the

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the Ca2+-gated RyRs and initiate Ca2+ sparks, as implied by ``local control theory''12,13 and indirectly inferred by previous experiments14±17. We therefore wanted to test directly whether a single LCC can trigger Ca2+ sparks, and if so, to determine the kinetics, ®delity and stoichiometry of the LCC±RyR coupling. When we applied the GQ-seal patch clamp and confocal imaging to myocytes with intact SR function (that is, in the absence of the SR inhibitors), we detected four Ca2+ sparks that were triggered unequivocally by iCa in 2 out of 28 patches that displayed LCC activities. Figure 3 shows that a large tail iCa (0.82 pA, 18.7 ms) on hyperpolarization from +30 mV to -110 mV evokes a typical Ca2+ spark with a latency of 4.2 ms. Notably, the LCC open duration in the presence of FPL outlasts the spark rise time (7 ms), suggesting that once triggered a Ca2+ spark evolves and terminates autonomously, independent of the duration of trigger iCa (ref. 14). As LCC openings did not trigger a Ca2+spark in most GQ-seal patches, we thought that membrane deformation associated with the GQ-seal might result in LCC±RyR uncoupling on most occasions. To preserve the functional integrity of the coupling apparatus, we implemented a `loose' patch-clamp technique; that is, a lowresistance (20±50 MQ) seal formed by gently pressing the patch pipette (5±7 MQ) against the surface membrane without suction (Fig. 4a, inset). Although resolution of subpicoampere iCa is beyond the theoretical limits set by the inverse relation between seal resistance and electrical noise7 in such loose patches, detection of Ca2+ sparklets would nonetheless enable us to monitor single LCC activity by optical means. In the presence of FPL, patch depolarization to ,0 mV evoked both high- and low-amplitude local Ca2+ events (Fig. 4a), giving rise to a bimodal amplitude distribution, with peaks around 20 and 100 nM, respectively (Fig. 4d, top panel). The low-amplitude events were resistant to 10 mM ryanodine, but were completely abolished by 2 mM nifedipine (Fig. 4a), and thus represent Ca2+ sparklets. When we decreased the pipette Ca2+ to 1 mM and omitted FPL, we could not detect a Ca2+ sparklet owing to a markedly reduced qCa. However, the high-amplitude events were still activated robustly (Fig. 4c), and exhibited a broad bell-shaped amplitude distribution similar to the ryanodine-sensitive population activated with 20 mM pipette Ca2+ (Fig. 4d). Hence, the high-amplitude events are Ca2+

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Figure 1 Confocal visualization of single LCC Ca2+ transients, `Ca2+ sparklets'. Top, simultaneous recordings of confocal line-scan image; middle, normalized ¯uo-4 ¯uorescence; bottom, single LCC currents (iCa). Inset, experimental setting. The patch membrane was held at -50 mV and depolarized for 400 ms to voltages between -40 mV and 0 mV at 0.1 Hz. The membrane potential outside the patch was zeroed by a NATURE | VOL 410 | 29 MARCH 2001 | www.nature.com

high K+/ 0 Ca2+ solution. Note that LCC openings are associated with Ca2+ sparklets in a 1:1 fashion. These sparklets are apparently brighter (higher DF/F0) than those shown in Figs 4 and 5, mainly because of a low resting [Ca2+], and thus a low F0, in the absence of extracellular Ca2+.

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Figure 3 A Ca2+ spark triggered by single LCC opening. The cell was bathed in normal physiological saline (see Methods). The resting potential (RP) in rat ventricular myocytes is around -70 mV (ref. 28). A Ca2+ spark is evoked by a tail iCa on hyperpolarization from RP +100 mV to RP -40 mV. Note that the spark declines before L-type-channel current switches off.

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®rst bin). The exponential distribution of coupling latency indicates that Ca2+ sparklets ignite Ca2+ sparks in a stochastic manner. Because of stochastic variations of both LCC open time and LCC±RyR coupling latency, not every Ca2+ sparklet would be expected to trigger a Ca2+ spark. We therefore determined the LCC±RyR coupling ®delity (d); that is, the fraction of sparklets that successfully trigger sparks. As shown in Fig. 6d, 71% of the ®rst sparklets on depolarization triggered Ca2+sparks, whereas 29% of them did not (that is, d1 = 0.71), indicating a probabilistic rather than deterministic nature of LCC±RyR coupling. When the ®rst sparklet did not trigger a spark, the conditional ®delity for the second sparklet remained unchanged (d01 = 0.67, P . 0.05 versus d1, x2-test). This suggests that, once terminated, a Ca2+ sparklet exerts no signi®cant effect on subsequent LCC±RyR coupling, as the local [Ca2+] gradient dissipates in a fraction of a millisecond18. When the ®rst sparklet did trigger a spark, however, the subsequent coupling ®delity was signi®cantly reduced (d11 = 0.30, P , 0.01 versus d1 or d01, x2-test), suggesting a use-dependent inactivation5,9 or ``adaptation''19 of RyRs in intact cells. A fundamental and unsettled issue regarding LCC±RyR coupling is the number of RyRs that are activated by a single LCC. In fact, whether a spark re¯ects a single RyR acting solo or a cluster of RyRs acting in concert remains a controversial issue6,20±25. Because sparklets and triggered sparks share common Ca2+ buffering and diffusion microenvironments, Ca2+ sparklets of known iCa would offer a natural standard for calibration of the Ca2+ ¯ux underlying a spark

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Figure 4 Sparklet±spark coupling under loose patch-clamp conditions. Inset, experimental setting. a, Top, both low-amplitude sparklets and high-amplitude sparks are evoked during patch depolarization from RP to ,0 mV. Middle, 10 mM ryanodine blocks Ca2+ sparks within ,3±5 min without affecting Ca2+ sparklet activity. Bottom, a subsequent application of 2 mM nifedipine, an LCC antagonist abolishes the remaining Ca2+ sparklets (bottom). b, Nifepidine alone (2 mM, ,3±5 min) abolishes both Ca2+ sparklet and the spark. c, Patch depolarization with Ba2+ as the charge carrier fails to evoke Ca2+ sparks; however, patch depolarization with 1 mM Ca2+ in the pipette in the 594

absence of FPL robustly evokes Ca2+ sparks. d, Amplitude histograms of local Ca2+ events under various experimental conditions. Top, intact SR with 10 mM FPL and 20 mM pipette Ca2+; middle, after ryanodine treatment; bottom, intact SR with 1 mM pipette Ca2+ without FPL. The broad modal distribution of spark amplitudes differs from the monotonic decaying distribution reported previously22, because the present experimental settings eliminate the `out-of-focus' events that have confounded previous confocal study of sparks6,21,22.

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letters to nature about 4±6 RyRs to generate a Ca2+ spark. In summary, we have developed the optical measurement of a single Ca2+ channel, and investigated the Ca2+ signalling between LCCs and RyRs at single-channel level. We have shown that a Ca2+ sparklet from a single LCC triggers SR Ca2+ release from four to six RyRs, directly validating the notion that cardiac SR Ca2+ release is under exquisite local control by single LCCs. Although Ca2+ sparklets tightly follow the open and close of LCCs, Ca2+ spark duration is relatively independent of the triggering sparklet. The LCC±RyR coupling exhibits a stochastic kinetics; the coupling ®delity is less than unity and use dependent. These ®ndings provide insights into

(jspark). By comparing the initial rising rate of the triggered sparks (n2, Fig. 6a), the sparklet foot (n1) or the solitary sparklet (n0), we found that local [Ca2+] rises about seven times faster during sparks than sparklets (n2/n0 = 7.5, n2/n1 = 6.7; Fig. 6c). Given an iCa amplitude of 0.30 pA at 0 mV, and assuming a linear relationship between the injecting Ca2+ ¯ux and the rising rate of local [Ca2+], we estimated the jspark to be ,2.1 pA. This value falls within the lower bound of model-derived estimation (2±10 pA)6,23,26, but is well above the unitary Ca2+ current of cardiac RyRs (,0.35±0.6 pA measured in planar lipid bilayers under quasi-physiological ionic conditions)27. Thus, our data suggest that an LCC typically triggers RP + 70 mV

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Figure 5 Visualization of the Ca2+ sparklets that immediately precede triggered Ca2+ sparks. Left, confocal images taken at a high temporal resolution (0.7 ms per line) and signal-to-noise ratio afforded by a LSM510 confocal microscope. The arrow indicates a

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Figure 6 Latency and ®delity of LCC±RyR coupling. a, Illustration showing the measurement of the initial rising rate of the sparklet foot (n1) and triggered spark (n2), and their coupling latency (L). n1 and n2 were determined by linear ®t of the ®rst eight data points (for events with L . 5.6 ms). b, Histogram of the sparklet±spark coupling latency. Solid curve represents the least-squares single-exponential ®t to the data. Time constant t = 6.7 ms. c, Rising rates for the solitary sparklets (n0, n = 14), the foot (n1, n = 19) and NATURE | VOL 410 | 29 MARCH 2001 | www.nature.com

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the upstroke (n2, n = 19) of triggered sparks. Note that n0 < n1 p n2. Asterisk, P , 0.01 versus n0 or n1. d, Fidelity of LCC±RyR coupling for the ®rst events on depolarization (d1), the second events when the ®rst sparklet did not trigger a spark (d01), and all events after a triggered spark (d11). Note that d1 < d01 . d11. Asterisk, P , 0.01 versus d1 or d01, x2test.

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letters to nature the molecular mechanisms of cardiac excitation±contraction coupling, and may have important implications for studying Ca2+ signalling in general. M

Methods Confocal Ca2+ imaging Enzymatically isolated adult rat ventricular myocytes were loaded with the Ca2+ indicator ¯uo-4-AM as described6,28. Confocal line-scan imaging was performed using a Zeiss LSM 410 or LSM510 confocal microscope equipped with an argon laser (488 nm) and a ´40, 1.3NA oil immersion objective. Line-scan images were acquired at sampling rates of 0.7 or 1.4 ms per line and 0.07 mm per pixel, with radial and axial resolutions of 0.4 and 1.0 mm, respectively. Digital image analysis was performed using IDL software (Research Systems) and customer-devised routines6,22. In SR-paralysed cells, local Ca2+ transients were indexed by the normalized local ¯uorescence, F/F0 (F0 refers to the level before depolarization). In cells with intact SR function, local Ca2+ transients were determined using the following formula6: ‰Ca2‡ Š ˆ kd R=…kd =‰Ca2‡ Šrest ‡ 1 2 R† where R = F/F0, the resting Ca2+ concentration [Ca2+]rest = 100 nM, and the dissociation constant kd = 1.1 mM (in accordance with the value for ¯uo-3 in cells29).

Cell-attached patch clamp Cell-attached patch clamps were established in either GQ-seal or loose-seal con®guration7, with glass patch pipettes of 5±7 MQ. In GQ-seal patches, unitary Ca2+ currents were recorded by using a cooled capacitor feedback headstage (CV203B) and an Axopatch 200 B ampli®er (Axon Instruments). Current records were low-pass ®ltered at 1 kHz, digitized at 5 kHz, and are presented with leak and capacity currents eliminated by subtraction of smooth functions, as described9. For loose-seal patch clamp experiments, membrane potential was determined by proportionally dividing the test voltages between the pipette resistance (5±7 MQ) and the seal resistance (20±50 MQ). The surface-charge shielding effect by high Ca2+ or Ba2+ was not corrected.

Solutions The standard patch pipette ®lling solution contained (in mM): 120 tetraethylammonium chloride, 20 CaCl2, 0.01 tetrodotoxin, 0.01 FPL64176 and 10 HEPES, pH 7.4, unless speci®ed otherwise. In experiments shown in Fig. 1, the extracellular solution outside the patch membrane contained (in mM): 100 potassium aspartate, 40 KCl, 10 HEPES, 1 EGTA, 5 Mg2+-ATP, 10 caffeine and 0.01 thapsigargin, pH 7.3. In other experiments, the standard superfusion solution contained (in mM): 135 NaCl, 1 CaCl2, 4 KCl, 1 MgCl2, 10 glucose, 10 HEPES, pH 7.4 . All experiments were performed at room temperature (23± 25 8C).

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Acknowledgements We thank W. J. Lederer, M. B. Cannell, M. D. Stern, E. RõÂos, J. S. K. Sham, S. J. Sollott, I. Josephson and R. P. Xiao for critical comments on the manuscript; H. A. Spurgeon for technical support; and A. Erauth for secretarial assistance. This work was supported by the NIH intramural research program (to E.G.L. and H.C.) and grants from the National Natural Science Foundation of China (H.C.). Correspondence and requests for materials should be addressed to H.C. (e-mail: [email protected]).

................................................................. Drought-induced guard cell signal transduction involves sphingosine-1-phosphate Carl K.-Y. Ng*, Kathryn Carr², Martin R. McAinsh*, Brian Powell² & Alistair M. Hetherington* * Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK ² Avecia Limited, Hexagon House, Blackley, Manchester M9 8ZS, UK ..............................................................................................................................................

Stomata form pores on leaf surfaces that regulate the uptake of CO2 for photosynthesis and the loss of water vapour during transpiration1. An increase in the cytosolic concentration of free calcium ions ([Ca2+]cyt) is a common intermediate in many of the pathways leading to either opening or closure of the stomatal pore2,3. This observation has prompted investigations into how speci®city is controlled in calcium-based signalling systems in plants. One possible explanation is that each stimulus generates a unique increase in [Ca2+]cyt, or `calcium signature', that dictates the outcome of the ®nal response4. It has been suggested that the key to generating a calcium signature, and hence to understanding how speci®city is controlled, is the ability to access differentially the cellular machinery controlling calcium in¯ux and release from internal stores2±5 . Here we report that sphingosine-1phosphate is a new calcium-mobilizing molecule in plants. We show that after drought treatment sphingosine-1-phosphate levels increase, and we present evidence that this molecule is

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