CaSR Regulates Nerve Terminals Figure 4. Kinetics and dose-dependence of Calindol inhibtion of NSCC currents in synaptosomes. A, contiguous plot of NSCC current amplitude versus time showing timecourse of on and off kinetics for Calindol action in a cell-attached synaptosome recording. The outward current elicited by voltage step (200 ms, 240 to 110 mV) every 5 seconds is plotted against time. NSCC current was reversibly and reliably activated by reductions in bath [Ca2+] (0.06–6 mM; blue trace) and inhibited by Calindol (0.1–10 mM; green trace). The Ca2+ and Calindol axes are logarithmic and the absence of the green trace indicates Calindol concentration is zero. Calindol (10 mM) inhibited NSCC current after a 1–2 minute delay and thereafter blocked with mono-exponential time course. The current increased with an exponential time course following reduction of Calindol. B, Calindol inhibited NSCC currents in bath [Ca2+] of 60 mM with an IC50 6.361.1 mM on average (n = 4). Each recording was normalized by dividing the NSCC current amplitude by the NSCC current amplitude elicited by 60 mM bath [Ca2+] before Calindol was applied. Calindol was generally applied at higher concentrations first and the amplitude of the NSCC current increased after washout (closed triangle) compared to initial baseline (open circle). This presumably reflects a run-up phenomenon due to the long duration of these experiments. C, Calindol inhibition of NSCC current is biphasic (i.e. latency and monoexponential) whereas Ca2+ inhibition is well described by a single exponential. Two sections of the timecourse data in A displaying applications of 10 mM Calindol and 6 mM Ca2+ (just prior to Calindol application) were redrawn on expanded time axis and overlaid so that time zero corresponded to solution change. Both datasets are well-fit by single exponentials with Calindol decaying significantly more slowly (tau of 73 s for Calindol vs 6.6 s for Ca2+) and at a substantial latency. doi:10.1371/journal.pone.0008563.g004 current activated by 60 mM Ca2+ in cell-attached recordings from synaptosomes (Figure S2). The high NPS-467 concentration required is consistent with other reports using expressed CaSR [40]. CaSR and mGluR1 are both family C GPCRs and although they only have an amino acid identity ,24% they are similar in terms of their large extracellular domains, tendency to function as dimers, and because they both possess a membranous binding PLoS ONE | www.plosone.org 5 January 2010 | Volume 5 | Issue 1 | e8563