Calcium (Ca²⁺) regulates various biological and pathological functions via calcium dynamics and interacting with key calcium binding proteins such as the calcium sensing receptor (CaSR). In this dissertation, the first X-ray structure of the extracellular domain of CaSR was determined by engineering mammalian expression systems. The revealed Ca²⁺/Mg²⁺ and Trp derivative L-1,2,3,4-tetrahydronorharman-3-carboxylic acid (TNCA) binding sites and key determinants contribute to the functional cooperativity of CaSR in cells. Magnesium (Mg²⁺) acts as a heterotropic cooperative co-agonist with calcium to co-activate the function of CaSR, including calcium oscillations. TNCA potentiates CaSR co-activation and recovers a loss of function caused by mutation at the dimer interface calcium binding site. Several mutations of the main Ca²⁺/TNCA binding site at the hinge region eliminate CaSR activity. Mutations S272A and D216N at the hinge region lead to a loss of Ca²⁺ binding and complete loss of cooperative binding for Tb³⁺ using bacterially expressed protein and Trp-sensitized FRET assay. Efforts in the development of new CaSR therapeutics using structure-based drug design were also explored.

Next, we aimed to monitor endoplasmic/sarcoplasmic reticulum (ER/SR) mediated subcellular Ca²⁺ dynamics using our designed calcium sensors CatchER⁺ and CatchER⁺-JP45. Using highly inclined laminated optical (HILO) microscopy, we report calcium dynamics in the ER/SR with differential calcium responses to 4-cmc for release and recovery indicating differential Ca²⁺ signaling from Ca²⁺ and protein expression subcellular microdomains. We find Ca²⁺ dynamic differences between the localized high Ca²⁺ release region of the junctional SR for E-C coupling with targeted CatchER⁺-JP45 to ryanodine receptor over the global Ca²⁺ ER/SR regulation of CatchER⁺ sensor. To understand ER Ca²⁺ dynamics in neurons, we utilized our sensor CatchER⁺ and high-resolution HILO imaging to show that 100 µM DHPG induced mGluR1/5 activation leads to IP₃R Ca²⁺ release as well as Ca²⁺ uptake throughout the soma and dendrites. The differential release and uptake for the ER Ca²⁺ dynamics in response to DHPG indicates subcellular microdomains throughout the neurons as well. These sensors will significantly impact Ca²⁺ dynamics research and molecular basis of ER Ca²⁺ related diseases by exposing Ca²⁺ dynamics, function, mobility, and trafficking in the ER/SR.