Monitoring Subcellular Calcium by Designed Calcium Sensors and the Calcium Sensing Receptor Structure and Function

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