Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals Wenyan Chen1, Jeremy B. Bergsman 1 , Xiaohua Wang 1 , Gawain Gilkey1, Carol-Rene´ e Pierpoint1, Erin A. Daniel1, Emmanuel M. Awumey2, Philippe Dauban3, Robert H. Dodd 3 , Martial Ruat4, Stephen M. Smith1* 1 Division of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, Oregon, United States of America, 2 Julius L. Chambers Biomedical/ Biotechnology Research Institute, North Carolina Central University, Durham, North Carolina, United States of America, 3 Centre National de la Recherche Scientifique, Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France, 4 Centre National de la Recherche Scientifique, Institut de Neurobiologie Alfred Fessard, Gif-sur- Yvette, France Abstract Background: Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca2+] ([Ca2+] ) indirectly regulates a non-selective cation channel (NSCC) in o neocortical nerve terminals via an unknown [Ca2+]o sensor. Here, we identify the first component in a presynaptic calcium signaling pathway. Methodology/Principal Findings: By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca2+]o than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents. Conclusions/Significance: Our experiments identify CaSR as the first component in the [Ca2+]o sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca2+]o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca2+]o following its entry via voltage-activated Ca2+ channels. CaSR may detects such falls in [Ca2+]o and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca2+]o on release probability. CaSR is positioned to detect the dynamic changes of [Ca2+]o and provide presynaptic feedback that will alter brain excitability. Citation: Chen W, Bergsman JB, Wang X, Gilkey G, Pierpoint C-R, et al. (2010) Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals. PLoS ONE 5(1): e8563. doi:10.1371/journal.pone.0008563 Editor: Olivier Jacques Manzoni, INSERM U862, France Received October 20, 2009; Accepted December 8, 2009; Published January 5, 2010 Copyright: 2010 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the OHSU-HHMI Biomedical Support Program, NINDS (R01-NS 43444), and OHSU President’s Fund. M.R. is supported by a grant from the French Agence Nationale de la Recherche (07-physio-027-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: R.H.D., P.D., and M.R. have filed a use-patent for Calindol. This does not alter adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: smisteph@ohsu.edu Introduction Neurotransmitter release from nerve terminals underlies synaptic communication in the brain. Invasion of the nerve terminal by an axonal spike activates voltage-gated channels, triggering calcium entry and exocytosis of transmitter-containing vesicles [1]. Release probability at a given synapse is dynamic; the ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry [2,3,4]. Characterization of the functional properties of ion channels at nerve terminals is thus crucial to understand presynaptic regulation of transmitter release. Extension of patch clamp techniques to small, relatively inaccessible nerve terminals has substantially increased our understanding of presynaptic function at these important sites [4,5]. One unexpected finding is that external [Ca2+] ([Ca2+]o) indirectly regulates a non-selective cation channel (NSCC) in the vast majority of neocortical nerve terminals via an unknown [Ca2+]o sensor [6]. However the mechanism by which [Ca2+]o exerts these effects is poorly understood. Recent studies have underlined the central role in regulation of neurotransmission of a number of calcium signaling pathways [1,7]. While synchronous, asynchronous and spontane- PLoS ONE | www.plosone.org 1 January 2010 | Volume 5 | Issue 1 | e8563