suggesting the previously isolated CaSR-mGluR and CaSR- GABABR heterodimers may operate in alternative subcellular compartments or in other brain regions. CaSR Is the Nerve Terminal [Ca2+]o Sensor We demonstrated that CaSR is present in nerve terminals by immunoblot and immunofluorescence, that [Ca2+]o has reduced potency at terminals from heterozygous mutant CaSR- containing mice, and that Calindol modulates terminal NSCC currents. These data strongly support the hypothesis that the mechanism by which changes in [Ca2+]o are transduced in neocortical nerve terminals involves CaSR. Moreover, the lack of any action of glutamate or GABA on the NSCC current indicate that neither mGluR nor GABABR act as neocortical nerve terminal [Ca2+] - o sensors that regulate the NSCC current. The [Ca2+]o-sensor-NSCC signaling pathway was present in the vast majority of neocortical nerve terminals from mouse (93%) and rat (79%) consistent with a pervasive role in neuronal signaling. The IC50 of Ca2+ inhibition of mouse neocortical terminal NSCC current (1.1 mM) was lower than the EC50 for Ca2+ activation of heterologously expressed CaSR (1.75–4.1 mM) [31,39,47,48]. There are a number of possible explanations for this apparent difference. First, both sets of measurements detected different functional changes downstream of CaSR and thus any non- linearities in the signaling pathways would change the apparent affinity for Ca2+. Second, desensitization has also been shown to alter the CaSR concentration-effect relationship and reduce the apparent affinity of Ca2+ [39]. Regulation of CaSR desensitization is not fully understood [39,49]. However, in our experiments in nerve terminals, evidence of desensitization was only present in experiments lasting .30 minutes (Figure 4); NSCC current amplitudes were generally stable over periods shorter than this (Figure 7B,E). This contrasted with the relatively transient nature of the rise of [Ca2+]i in CaSR expressing HEK cells (Figure 5) and may in part explain differences in apparent affinities between preparations. Third, the discrepancies in affinity may arise from differences between expression systems and native tissue receptors resulting from post-translational modification or protein-protein interactions. Consistent with this an increased affinity has been reported for CaSR signaling in glial cells [50]. The IC50 of [Ca2+]o inhibition of terminal NSCC current shifted from 1.1 to 1.6 mM (Figure 1; CaSR+/+ and CaSR+/2 respectively). This modest shift in affinity is in line with the changes reported following heterologous co-expression of normal and mutant CaSR [31] and may be attributed to partial rescue of CaSR mutants by wild- type receptor (Wang and Smith, unpublished observations). The small difference in affinity is reflects the mildness of the phenotype of the heterozygote mouse; the serum ionized calcium levels in CaSR+/+ and CaSR+/2 mice are 1.260.1 and 1.460.0 mM, respectively [30]. Latency of Calindol Action Calindol is an allosteric agonist that binds to a relatively inaccessible membranous pocket on CaSR, increasing the sensitivity of the receptor to Ca2+ [36]. The striking difference between both agonists in the time to onset of action (Figs. 4C,5B) presumably reflects differences in the mechanism of action of Ca2+ and Calindol. The much longer latency for Calindol may reflect the time taken for it to reach and bind to the less accessible membranous pocket and/or the time taken for CaSR to alter conformation once Calindol is bound. Inhibition of NSCC currents by Calindol exhibited a slower exponential rate compared to when Ca2+ was applied at 6 mM (Figure 4C). As the bath [Ca2+] was 60 mM during the Calindol application, the ten-fold CaSR Regulates Nerve Terminals decrease in the rate of NSCC current block may be postulated to reflect the reduced rate of Ca2+ binding to CaSR. However this explanation is unsatisfactory as Ca2+-mediated NSCC current block was more rapid than Calindol-mediated block even when much lower [Ca2+]o was employed (Figures 2C & 7B,E). An alternative explanation is that the second phase (exponential decline) of NSCC current block by Calindol may in part be attributed to conformational changes. Further experiments are required to test these hypotheses. The latency for Calindol was smaller in CaSR-transfected HEK cells than in synaptosomes and may reflect differences in the signaling downstream of CaSR. Heterodimers CaSR exists as homodimers which form in the endoplasmic reticulum before transportation to the cell membrane [51]. Heterodimers comprised of CaSR and other GPCRs have also been identified [27,52]. Howevere these data make it unlikely that Ca2+-modulated currents in rat nerve terminals are mediated by heterodimers composed of CaSR and mGluR or GABABR as CaSR-mGluR heterodimers retain sensitivity to glutamate [27]. Our data do not exclude the possibility that CaSR is forming heterodimers with other GPCRs. GPRC6A, a recently identified group C GPCR, is one potential candidate that may hetero- dimerize with CaSR based on similarities in structure and sensitivity to [Ca2+]o [53,54]. Function of Nerve Terminal CaSR CaSR is associated with epilepsy [55] and dementia [56] however the consequences of CaSR activation in the CNS have not been fully characterized. In contrast, CaSR has been intensively studied in the periphery due to its central role in systemic calcium homeostasis [57]. CaSR activation by external Ca2+ regulates parathyroid hormone and calcitonin secretion from the parathyroid and thyroid glands, respectively, and thus maintains serum [Ca2+] [58,59,60]. Brain extracellular [Ca2+] is dynamic and decreases as neuronal activity increases- moderate decreases in [Ca2+]o accompany bursts of synaptic activity [61] while profound falls to ,0.1 mM result from focal brain trauma or ischemia [62,63]. These falls in [Ca2+]o will reduce stimulation of CaSR and thereby increase NSCC activity in the vast majority of neocortical nerve terminals [28]. Our current model proposes that decreases in cleft [Ca2+] will reduce Ca2+ entry and synaptic transmission [8] but that the fall in cleft [Ca2+] will act as feedback to presynaptic CaSR. Increased NSCC activity may increase action potential duration [64], prolong Ca2+ entry at the terminal and thus increase release probability [65]. Consequently CaSR may be operating as part of a homeostatic pathway to prevent synaptic failure when [Ca2+]o falls [28]. Alterations of such a homeostatic pathway could lead to increased excitability and gain-of-function CaSR mutations have been associated with childhood epilepsy [66]. One possible explanation is that the increased calcium affinity of gain-of- function CaSR mutants may steepen the response to normal changes in [Ca2+]o, lead to overcompensation of the homeostatic pathway and increase the probability of aberrant activity. Consistent with this, loss-of-function CaSR2/2 mutant neurons have a more negative resting membrane potential [28] which could reduce the likelihood of seizures. However, the CaSR2/2 mutation increases release probability at excitatory synapses at basal [Ca2+]o [28] which is expected to increase neuronal excitability and increase the risk of epilepsy. Clearly additional study is required to determine how CaSR mutations lead to epilepsy [55]. PLoS ONE | www.plosone.org 8 January 2010 | Volume 5 | Issue 1 | e8563