Articles by Rakshya Gorkhali in JoVE
Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ Florence N. Reddish1, Cassandra L. Miller1, Rakshya Gorkhali1, Jenny J. Yang1 1Department of Chemistry, Center of Diagnostics and Therapeutics (CDT), Georgia State University We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.
Other articles by Rakshya Gorkhali on PubMed
Defining Potential Roles of Pb(2+) in Neurotoxicity from a Calciomics Approach Metallomics : Integrated Biometal Science. Jun, 2016 | Pubmed ID: 27108875 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., Pb(2+)) involves disruption of synaptic activity, and these observed effects are associated with the ability of Pb(2+) to interfere with Zn(2+) and Ca(2+)-dependent functions. However, the molecular mechanism behind Pb(2+) toxicity remains a topic of debate. In this review, we first discuss potential neuronal Ca(2+) binding protein (CaBP) targets for Pb(2+) 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 Ca(2+)-signalling pathways. We then compare metal binding properties between Ca(2+) and Pb(2+) to understand the structural implications of Pb(2+) binding to CaBPs. Statistical and biophysical studies (e.g., NMR and fluorescence spectroscopy) of Pb(2+) binding are discussed to investigate the molecular mechanism behind Pb(2+) toxicity. These studies identify an opportunistic, allosteric binding of Pb(2+) to CaM, which is distinct from ionic displacement. Together, these data suggest three potential modes of Pb(2+) activity related to molecular and/or neural toxicity: (i) Pb(2+) can occupy Ca(2+)-binding sites, inhibiting the activity of the protein by structural modulation, (ii) Pb(2+) can mimic Ca(2+) in the binding sites, falsely activating the protein and perturbing downstream activities, or (iii) Pb(2+) can bind outside of the Ca(2+)-binding sites, resulting in the allosteric modulation of the protein activity. Moreover, the data further suggest that even low concentrations of Pb(2+) can interfere at multiple points within the neuronal Ca(2+) signalling pathways to cause neurotoxicity.
Structural Basis for Regulation of Human Calcium-sensing Receptor by Magnesium Ions and an Unexpected Tryptophan Derivative Co-agonist Science Advances. May, 2016 | Pubmed ID: 27386547 Ca(2+)-sensing receptors (CaSRs) modulate calcium and magnesium homeostasis and many (patho)physiological processes by responding to extracellular stimuli, including divalent cations and amino acids. We report the first crystal structure of the extracellular domain (ECD) of human CaSR bound with Mg(2+) and a tryptophan derivative ligand at 2.1 Å. The structure reveals key determinants for cooperative activation by metal ions and aromatic amino acids. The unexpected tryptophan derivative was bound in the hinge region between two globular ECD subdomains, and represents a novel high-affinity co-agonist of CaSR. The dissection of structure-function relations by mutagenesis, biochemical, and functional studies provides insights into the molecular basis of human diseases arising from CaSR mutations. The data also provide a novel paradigm for understanding the mechanism of CaSR-mediated signaling that is likely shared by the other family C GPCR [G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor] members and can facilitate the development of novel CaSR-based therapeutics.
Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor Frontiers in Physiology. 2016 | Pubmed ID: 27746744 Ca(2+)-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca(2+)]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 Ca(2+), Mg(2+), 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 Ca(2+) 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.