ous transmitter release have all been shown to strongly depend on extracellular Ca2+ [7,8,9] these effects are usually attributed to Ca2+ entry via voltage-activated Ca2+ channels (VACC) and less attention has been focused on the presynaptic role of other Ca2+ signaling pathways, such as surface charge screening [10,11,12] or Ca2+-dependent ion channels [6,13,14]. Attention is returning to these other pathways with the realization that their influence may have been underappreciated because supraphysiological [Ca2+]o employed in most studies ensured maximal receptor activation and decreased the impact of physiological decreases in external [Ca2+]. In this study we identify the receptor activating a novel calcium signaling pathway using direct patch clamp recordings from nerve terminals. Candidate [Ca2+]o receptors include the extracellular calcium-sensing receptor (CaSR), metabotropic glutamate recep- tor (mGluR) and c-aminobutyric acid B receptor (GABABR). All of these receptors have been identified as sensitive to [Ca2+]o [15,16,17,18], have been localized to the synapses of central neurons [19,20,21,22,23], and have been classified as members of G-protein coupled receptor (GPCR) family C [24]. In addition, CaSR may heterodimerize with mGluR and GABABR [25,26,27], raising the possibility that heterodimers involving some or all of these GPCRs may modulate NSCC currents in nerve terminals. Our experiments, studying the impact of CaSR agonists and a CaSR mutation on the [Ca2+]o sensor-NSCC pathway in nerve terminals, show that the calcium-sensing receptor (CaSR), the mainstay in serum calcium homeostasis, is the extracellular calcium sensor regulating NSCC activity in neocortical nerve terminals. In contrast, nerve terminals were insensitive to glutamate and GABA arguing strongly against the mGluR and the GABABR mediating these effects. This approach provides insight into a novel pathway through which [Ca2+]o influences nerve terminal excitability. Results CaSR Mutation Reduces Affinity of [Ca2+]o Detector Small, acutely isolated neocortical nerve terminals sense [Ca2+]o and indirectly modulate a NSCC current (Figure 1A) as reported previously [6,28]. The CaSR2/2 mutant mouse lacks CaSR exon 5 which results in a reduced affinity for Ca2+ [29]. However, CaSR2/2 mutant mice die prematurely and exhibit delayed growth, preventing the preparation of synaptosomes from these animals. We therefore used the heterozygous mouse (CaSR+/2), which has an elevated serum [Ca2+] reflecting lower affinity for [Ca2+]o but normal growth and survival [30], to examine if the CaSR mutation impacts nerve terminal sensitivity to [Ca2+]o. In cell-attached recordings, 71 of 76 mouse neocortical nerve terminals (93%) possessed the characteristic [Ca2+]o-modulated outward current. Outward NSCC currents were activated by depolarization from 240 mV to 110 mV (all voltages were relative to the resting membrane potential) while [Ca2+]o was changed between 6 mM and 60 mM (Figure 1A,B). Outward currents were larger at lower [Ca2+] . The CaSR+/2 terminals o were less sensitive to increases in [Ca2+]o as illustrated by the traces activated with 0.6 and 6 mM Ca2+ in the bath solution (Figure 1A– C). The activation kinetics (Figure 1A,B) were similar for CaSR+/+ and CaSR+/2 terminals. The concentration-effect relationship for the normalized NSCC current amplitudes (Figure 1D) confirmed that CaSR+/2 terminals had a lower affinity than the CaSR+/+ terminals (IC50 1.660.2 mM versus 1.160.07 mM respectively; ANOVA, p = 0.032). We measured the NSCC current amplitudes elicited by 6 mM and 60 mM bath Ca2+ to test if the maximum and minimum currents respectively were also dependent on genotype. However, CaSR+/+ and CaSR+/2 terminals had similar CaSR Regulates Nerve Terminals maximum (2065 pA, n = 16 vs 1764 pA, n = 13; p = 0.61) and minimum currents (24.563.5 pA, n = 7 vs 23.362.1 pA; p = 0.79) indicating the IC50, but not NSCC current amplitude was dependent on the CaSR genotype. The reduction in affinity for [Ca2+]o in CaSR+/2 terminals was consistent with CaSR involvement in modulation of the NSCC currents. The relatively modest shift in affinity is similar to the changes observed following heterologous co-expression of normal and other mutant CaSR [31]. CaSR Is Present in Nerve Terminals We next tested that CaSR was present in neocortical nerve terminals using immunochemical techniques. CaSR was present in rat whole brain tissue and synaptosomes by immunoblotting. Western blots detected 140 and 160 kDa bands in HEK cells transfected with CaSR (Figure 2A, left lane). These have been shown to represent differentially glycosylated forms [29] and were absent in untransfected control cells (Figure 2A, second lane). Synaptosomes and whole brain (Figure 2A, third and fourth lanes, respectively) contained the 160 kDa band and a lower band at 90 kDa, both of which were absent in control experiments following preincubation with the specific antigenic peptide fragment (data not shown). The 90 kDa band which has been reported by others was also blocked by peptide fragment and appears to be due to CaSR degradation [32]. To confirm CaSR was present in nerve terminals we used a polyclonal antibody raised against CaSR (4641; Figure 2C) and co-stained synapto- somes with an antibody to synaptophysin (Figure 2B). Both antibodies gave similar punctate patterns of staining that co- localized (Figure 2D) indicating that CaSR is indeed present in neocortical nerve terminals. The polyclonal antibody 4641 also identified the 140 and 160 kDa bands in Western blots consistent with a specific action (data not shown). The Nerve Terminal [Ca2+]o Sensor Is Modulated by Allosteric CaSR Agonists CaSR agonists including Ca2+, Mg2+, spermidine, gadolinium, and neomycin have been shown to modulate NSCC currents in nerve terminals [6,33]. However, since these CaSR agonists also interact with other targets [34], we employed more specific pharmacological interventions against CaSR to explore the identity of [Ca2+]o-modulated NSCC. Allosteric CaSR agonists bind to a transmembrane pocket of CaSR [35,36]. We tested if the [Ca2+]o-modulated NSCC currents in rat synaptosomes were sensitive to Calindol, an allosteric CaSR agonist [37]. Out of 290 cell-attached recordings from rat neocortical nerve terminals, 230 (79%) possessed the characteristic [Ca2+]o-modulated outward current. In the cell-attached configuration, NSCC currents activated by depolarization with a [Ca2+]o of 60 mM were substantially and reversibly inhibited by bath application of 10 mM Calindol (Figure 3A). Control experiments showed that the solvent (ethanol) had no effect on the NSCC current amplitude at 0.1% (Figure S1). Calindol acted in an apparently allosteric fashion shifting the concentration-effect relationship for [Ca2+]o to the left. The average data from 11 recordings (Figure 3B) revealed a decrease in slope as well as a left-shift with 2 mM Calindol. To test whether the effect of Calindol was reduced in the presence of very low [Ca2+] as expected for an allosteric agonist, we reduced the bath [Ca2+] to nominally 0.2 mM and patch pipette solution [Ca2+] and [Mg2+] from 2 mM to 100 mM. Under these conditions, Calindol did not affect the NSCC currents (Figure 3C; control versus test: 4.562.4 pA versus 4.562.6 pA control versus; p.0.05, n = 4). These findings indicate that Calindol modulation of the [Ca2+]o-modulated NSCC signaling PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8563