Structural and functional characterization of the extracellular domain of the human CaSR with bound Mg2+ and a tryptophan derivative.

Metal ions play crucial roles in numerous biological processes, facilitating biochemical reactions by binding to various proteins. An increasing body of evidence suggests that neurotoxicity associated with exposure to nonessential metals (e.g., Pb2+) involves disruption of synaptic activity, and these observed effects are associated with the ability of Pb2+ to interfere with Zn2+ and Ca2+-dependent functions. However, the molecular mechanism behind Pb2+ toxicity remains a topic of debate. In this review, we first discuss potential neuronal Ca2+ binding protein (CaBP) targets for Pb2+ such as calmodulin (CaM), synaptotagmin, neuronal calcium sensor-1 (NCS-1), N-methyl-D-aspartate receptor (NMDAR) and family C of G-protein coupled receptors (cGPCRs), and their involvement in Ca2+-signalling pathways. We then compare metal binding properties between Ca2+ and Pb2+ to understand the structural implications of Pb2+ binding to CaBPs. Statistical and biophysical studies (e.g., NMR and fluorescence spectroscopy) of Pb2+ binding are discussed to investigate the molecular mechanism behind Pb2+ toxicity. These studies identify an opportunistic, allosteric binding of Pb2+ with CaM that is distinct from ionic displacement. Together, this data suggests three potential modes of Pb2+ activity related to molecular and/or neural toxicity: (i) Pb2+ can occupy Ca2+-binding sites, inhibiting the activity of the protein by structural modulation, (ii) Pb2+ can mimic Ca2+ in the binding sites, falsely activating the protein and perturbing downstream activities, or (iii) Pb2+ binds outside of the Ca2+-binding sites, resulting in allosteric modulation of the proteins activity. Moreover, the data further suggest that even low concentrations of Pb2+ can interfere at multiple points within the neuronal Ca2+ signalling pathways to cause neurotoxicity.

Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids, and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR's cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

The flow of intracellular calcium (Ca2+) is critical for the activation and regulation of important biological events that are required in living organisms. As the major Ca2+ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca2+ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca2+ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2+ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events.

Intracellular calcium (Ca) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.