Author ORCID Identifier

0000-0003-0546-3138

Date of Award

Summer 8-13-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny J. Yang

Second Advisor

Ning Fang

Third Advisor

Ming Luo

Fourth Advisor

Angela Mabb

Abstract

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.

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