Fluo-4 respectively. Briefly, cells were loaded for 30 min at 37uC with 2 mM AM X-rhod-1 or 5 mM AM Fluo-4 in Tyrode solution, and then placed on the stage of an inverted microscope (IX-70, Olympus). Images were acquired using a 1.2 N.A. 63x water immersion or PlanApo 1.42 N.A. 60x oil-immersion objective and cooled CCD camera (Orca-ER, Hamamatsu) with computer controlled shutter (UNIBLITZ VMM-D1, Vincent Associates, Rochester, NY). Fluorophores were excited with a halogen lamp. Fluo-4 was excited at 470–490 nm and fluorescence emission measured via a LP filter 510 nm while X-rhod-1 was excited at 542–582 nm and emission measured at 604–644 nm (TXRED- 4040B-OMF-ZERO, Semrock, NY). Images were captured and processed using Wasabi image software. The changes in [Ca2+]i are reported as fluorescence ratios (F/F0) where F0 represents the baseline fluorescence. About 10–20 cells with similar cell surface GFP fluorescence from a single field were analyzed simultaneously using Wasabi software. Data Analysis Data were analyzed using custom macros written in Igor Pro (Wavemetrics, Lake Oswego, OR). As previously dose-response data were fit with the Hill equation where I represented current at concentration A of agonist and Imax was the maximum current and n represented the Hill coefficient [77]. NSCC current amplitudes were measured over the last 2–5 ms of a 200 ms depolarizing step. In synaptosome recordings, latency or delay of action of Calindol was measured from the plot of NSCC current amplitude versus time (diary plot) using the interception of the line representing average NSCC current amplitude at 60 mM Ca2+ and the extrapolated exponential fitted to the NSCC current during inhibition by Calindol (see Figure 4C). The delay equaled the difference between the intercept and the time at which solutions were switched (t = 0). In experiments using HEK cells the responses were not described by single exponentials and so a different approach was taken. The standard deviation of F0 was measured and latency was defined as the time between solution change and the point at which F/F0 first deviated above F0 by more than four standard deviations. Tests for paired or unpaired replicates were used as appropriate. Mean6S.E.M. values are reported in general. In the case of nonparametric data, the Mann-Whitney test was used. All tests were two-tailed and a p value,0.05 was considered significant. Graphpad Prism software was used to calculate p-values. References 1. Neher E, Sakaba T (2008) Multiple roles of calcium ions in the regulation of neurotransmitter release. Neuron 59: 861–872. 2. Bischofberger J, Geiger JR, Jonas P (2002) Timing and efficacy of Ca2+ channel activation in hippocampal mossy fiber boutons. J Neurosci 22: 10593–10602. 3. Li L, Bischofberger J, Jonas P (2007) Differential Gating and Recruitment of P/ Q-, N-, and R-Type Ca2+ Channels in Hippocampal Mossy Fiber Boutons. Journal of Neuroscience 27: 13420–13429. 4. Geiger JR, Jonas P (2000) Dynamic control of presynaptic Ca(2+) inflow by fast- inactivating K(+) channels in hippocampal mossy fiber boutons. Neuron 28: 927–939. 5. Engel D, Jonas P (2005) Presynaptic Action Potential Amplification by Voltage- Gated Na+ Channels in Hippocampal Mossy Fiber Boutons. Neuron 45: 405–417. 6. Smith SM, Bergsman JB, Harata NC, Scheller RH, Tsien RW (2004) Recordings from single neocortical nerve terminals reveal a nonselective cation channel activated by decreases in extracellular calcium. Neuron 41: 243–256. 7. Xu J, Pang ZP, Shin OH, Sudhof TC (2009) Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nat Neurosci 12: 759–766. 8. Dodge F Jr, Rahamimoff R (1967) Co-operative action of calcium ions in transmitter release at the neuromuscular junction. Journal of Physiology 193: 419–432. 9. Goda Y, Stevens CF (1994) Two components of transmitter release at a central synapse. Proc Natl Acad Sci U S A 91: 12942–12946. 10. Armstrong CM, Cota G (1991) Calcium ion as a cofactor in Na channel gating. Proc Natl Acad Sci U S A 88: 6528–6531. CaSR Regulates Nerve Terminals Supporting Information Figure S1 NSCC current amplitudes were unaffected by ethanol. Plot of average normalized NSCC amplitude evoked by depolarizing steps (240 to 110 mV for 200 ms) in the presence of 60 mM bath Ca2+ from four recordings. Application of ethanol at 0.1% (black horizontal bar) was associated with a small (,10%), transient (,20 ms) decrease in NSCC current amplitude. At 200 seconds there was no detectable change in the NSCC. Found at: doi:10.1371/journal.pone.0008563.s001 (0.12 MB EPS) Figure S2 NPS-467 reversibly inhibits NSCC currents in nerve terminal recordings. NSCC currents activated by step depolariza- tions (240 to 110 mV relative to membrane potential) in the presence of 60 mM [Ca2+]o were decreased by co-application of 10 mM NPS-467. Each trace represents an average of 10–20 currents at steady state solution conditions from the same synaptosome-attached recording. Timecourse of NSCC current amplitude from the same recording at three [Ca2+]o (upper trace) revealed that NPS reversibly inhibited the NSCC current amplitude. Found at: doi:10.1371/journal.pone.0008563.s002 (0.28 MB EPS) Figure S3 The [Ca2+]o-modulated NSCC current in rat nerve terminals was unaffected by 2 mM glutamate. NSCC currents activated by step depolarizations (240 to 110 mV relative to membrane potential) over a range of bath [Ca2+]o (60 mM-6 mM, black traces) superimposed with those recorded in the presence of 2 mM glutamate (red traces). Each trace represents average of 10– 20 currents at steady state solution conditions from the same synaptosome-attached recording. Found at: doi:10.1371/journal.pone.0008563.s003 (0.44 MB EPS) Acknowledgments We thank Mr N. Vyleta and Drs K. Khodakhah, R. Zorec and M.T. Harnett for comments on the manuscript, Drs. J.G. Seidman and D. Connor for providing the CaSR mutant mice, Dr Julie Saugstad for providing the mGluR transfected cell-line and Drs K. Rodland and E. Nemeth for providing antibodies. Author Contributions Conceived and designed the experiments: JB SMS. Performed the experiments: WC JB XW GG CRP EAD SMS. Analyzed the data: WC XW GG CRP EAD SMS. Contributed reagents/materials/analysis tools: EA PD RHD MR. Wrote the paper: JB RHD MR SMS. 11. Frankenhaeuser B, Hodgkin AL (1957) The Action of Calcium on the Electrical Properties of Squid Axons. J Physiol 137: 218–244. 12. Hille B (2001) Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates. 13. Immke DC, McCleskey EW (2003) Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade. Neuron 37: 75–84. 14. Xiong Z, Lu W, MacDonald JF (1997) Extracellular calcium sensed by a novel cation channel in hippocampal neurons. Proceedings of the National Academy of Sciences of the United States of America 94: 7012–7017. 15. Wise A, Green A, Main MJ, Wilson R, Fraser N, et al. (1999) Calcium sensing properties of the GABA(B) receptor. Neuropharmacology 38: 1647–1656. 16. Kubo Y, Miyashita T, Murata Y (1998) Structural basis for a Ca2+-sensing function of the metabotropic glutamate receptors. Science 279: 1722– 1725. 17. Saunders R, Nahorski SR, Challiss RA (1998) A modulatory effect of extracellular Ca2+ on type 1alpha metabotropic glutamate receptor-mediated signalling. Neuropharmacology 37: 273–276. 18. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, et al. (1993) Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 366: 575–580. 19. Wittmann M, Marino MJ, Bradley SR, Conn PJ (2001) Activation of Group III mGluRs Inhibits GABAergic and Glutamatergic Transmission in the Substantia Nigra Pars Reticulata. Journal of Neurophysiology 85: 1960–1968. PLoS ONE | www.plosone.org 10 January 2010 | Volume 5 | Issue 1 | e8563