Figure 3. Calindol facilitates inhibition of NSCC currents by extracellular Ca2+. A, exemplar traces show NSCC currents reversibly inhibited by addition of Calindol (10 mM) to bath solution. Traces were recorded in cell-attached mode following 200 ms step depolarization with 60 mM Ca2+ and 0 Mg2+ in the bath and 2 mM Ca2+ and 2 mM Mg2+ in the pipette solution (inset). The substantial inhibition of NSCC current was reversed following washout (gray trace). B, the Ca2+ concentration-effect relationship was left-shifted by the allosteric CaSR agonist Calindol (2 mM). These data represent 11 synaptosome recordings, each normalized to the current observed in 6 mM Ca2+. C, average traces show NSCC currents unaffected by the addition of 10 mM Calindol (gray trace) to bath solution containing reduced [Ca2+] . o Traces were recorded in cell-attached recording following 200 ms step depolarization with 0.2 mM Ca2+ and 0 Mg2+ in the bath and 0.1 mM Ca2+ and 0.1 Mg2+ in the pipette solution (inset). Recordings were less stable at low divalent concentrations; traces are thus averages of 8 currents elicited with a 5 second duty cycle. doi:10.1371/journal.pone.0008563.g003 CaSR Regulates Nerve Terminals pathway in nerve terminals is allosteric, similar to its action on CaSR in heterologous expression systems [38]. Calindol Activation of Terminal CaSR Exhibits Substantial Delay We next examined the potency and kinetics of action of Calindol on the NSCC currents in synaptosomes. Cell-attached recordings were made from synaptosomes and the membrane depolarized from 240 to 110 mV every 5 seconds. In the exemplar recording, the current activated by a 200 ms depolar- ization briskly and reliably increased to ,6 pA following a decrease in bath [Ca2+] (blue line) from 6 mM to 60 mM (Figure 4A). Addition of Calindol (10 mM), denoted by the green line, slowly decreased the NSCC current amplitude seen with 60 mM Ca2+ in the bath. Subsequent decreases in Calindol (green line; range 0.1 to 10 mM) resulted in increases in NSCC current amplitude at a fixed bath [Ca2+] (blue line). Like the initial decrease in response to Calindol, the other changes in NSCC current amplitude were well described by exponential time courses. The average IC50 for Calindol was 6.361.1 mM at 60 mM bath [Ca2+] (n = 4, Figure 4B). The NSCC current amplitude tended to ‘‘run-up’’ during these prolonged recordings and consequently the NSCC current amplitudes for the wash (solid triangle) and the lower doses of Calindol were slightly larger than the control currents (Figure 4B). Closer inspection of the NSCC current-time plots (Figure 4C) shows the kinetics for the Calindol-induced and Ca2+-induced reductions in current amplitude were different. The expanded and superimposed time scales illustrate that the response to Ca2+ was much faster than the response to Calindol (Figure 4C). Moreover, inhibition of the NSCC current by Calindol occurred after a substantial delay (75 sec) which sharply contrasted with rapid onset of inhibition of NSCC currents when Ca2+ was increased from 60 mM to 6 mM. Calindol has not previously been reported to exhibit any delay or latency of action. If Calindol’s action on the [Ca2+]o-modulated NSCC signaling pathway in small nerve terminals was mediated via CaSR we reasoned that this latency should also be present in measurements using heterologously expressed CaSR. We tested this idea by studying CaSR modulation in transiently transfected HEK cells. In this expression system stimulation of CaSR activates phospholipase C, increasing inositol triphosphate production and release of Ca2+ from intracellular stores [39]. Transfected cells were identified via fluorescence of cotransfected EGFP. Changes in [Ca2+]i were measured via the increase in X-rhod1 fluorescence above baseline (F/F0). Increasing bath [Ca2+] from 1 to 5 mM (at t = 0) resulted in an average increase in F/F0 of 0.40060.017 (n = 12; individual and average responses represented by black and red traces respectively). After a five minute wash in 1 mM Ca, application of Calindol (10 mM) resulted in a F/F0 rise of 0.26960.017 in the same 12 cells (Figure 5A). Superimposing the time courses of effects shows that the response to Calindol occurs after a substantially larger lag (Figure 5B) than that observed after activation by Ca2+. Measurement of the latency (by deflection .4 S.D. above baseline noise) showed a substantial increase with Calindol compared to 5 mM Ca2+ (9.060.2 s vs 23.761.2 s, n = 22; p,0.001) in heterologously expressed CaSR consistent with our observation at isolated small terminals. Previously, NSCC currents in isolated nerve terminals have been reported to be insensitive to the weak CaSR agonist NPS-467 [6]. In light of the latency of action of Calindol (Figure 4C) we re- tested whether NPS-467 was effective on nerve terminals using longer applications and higher agonist concentrations. Application of NPS-467 (10 mM) inhibited 3465% (n = 4) of the NSCC PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8563