CaSR Regulates Nerve Terminals Figure 1. Loss of function CaSR mutation reduces NSCC current sensitivity to [Ca2+]o. Cell- attached recordings were made from (A) CaSR+/+ and (B) CaSR+/2 terminals and the Ca2+ in the bath solution applied to terminals varied between 6 mM and 60 mM as indicated. Step depolarizations (240 to 110 mV relative to resting membrane potential) were made every 5 seconds. Average current traces (n = 8215) are shown for each bath [Ca2+] at steady-state for two exemplar recordings. Note that outward currents elicited with 0.6 and 6 mM Ca2+ were proportionately larger in the heterozygote than in the wild-type recording. C) timecourse of NSCC current amplitude (measured at the end of the depolarizing step) in the same CaSR+/+ (filled circles, left axis) and CaSR+/2 (open circles, right axis) terminals as bath [Ca2+] was increased (upper trace). Steady state amplitude was reached in 5–10 s for both CaSR+/+ and CaSR+/2 genotypes. Axes were scaled to span the current amplitudes measured between with bath [Ca2+] between 6 mM and 60 mM. D, the concentration-effect relationship for both CaSR genotypes shows that wild-type terminals exhibited higher affinity for Ca2+ (p = 0.032). NSCC currents were normalized for each terminal by measuring the difference between the NSCC current and the 60 mM Ca2+-elicited NSCC current and dividing this by the difference between the NSCC currents elecited by 6 mM and 60 mM Ca2+. The curves represent mean6SEM of 7 and 6 recordings for CaSR+/+ and CaSR+/2, respectively. The curves were fit to the average data points resulting in Hill coefficients of 0.77 for both genotypes and IC50s of 1.660.2 mM and 1.160.07 mM for CaSR+/2 and CaSR+/+, respectively. doi:10.1371/journal.pone.0008563.g001 Figure 2. CaSR is present in nerve terminals of neocortex. A, immunoblot of synaptosomes and whole brain show 160 kDa bands with anti- CaSR antibody [76]. Positive control (HEK CaR) shows 140 and 160 kDa bands (glycosylated and unglycosylated forms) in CaSR- transfected HEK cells and no signal in untransfected HEK cells (HEK Con). B, acutely isolated nerve terminals (synaptosomes) identified using the synaptophysin antibody (red). C, CaSR identified with polyclonal antibody ‘‘4641’’ (green). D, superimposition of B and C shows that CaSR and synaptophysin are co-localized. doi:10.1371/journal.pone.0008563.g002 PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8563