Date of Award

5-4-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Dr. Jenny J. Yang

Second Advisor

Dr. Giovanni Gadda

Third Advisor

Dr. Donald Hamelberg

Fourth Advisor

Dr. Angela Mabb

Abstract

As a first and second messenger, calcium (Ca2+) regulates numerous biological processes. Its homeostasis is modulated by intracellular Ca2+ stores, membrane channels, pumps, and/or receptors (etc. calcium sensing receptor (CaSR)). To integrate multi-level Ca2+ dynamics, which exhibit a wide range of temporal kinetics, there is a pressing need to integrate Ca2+ signaling intracellularly and extracellularly, using a novel class of genetically encoded Ca2+ indicators (GECIs) with rapid kinetics and fully understanding the molecular mechanism of CaSR, respectively.

In the present study, we first report a green Ca2+ indicator called “G-CatchER+” that specifically reports rapid local ER Ca2+ dynamics. G-CatchER+ exhibits a superior Ca2+ on rate than G-CEPIA1er. G-CatchER+ also reports agonist/antagonist triggered Ca2+ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, RyRs, and SERCAs. Transgenic expression of G-CatchER+ in Drosophila muscle demonstrates its utility in stimulus-evoked SR local Ca2+ dynamics.

Second, we developed a red Ca2+ indicator based on mApple. R-CatchER showed superior kinetics in vitro, and in multiple cell types. It captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints. It also enabled the first report of ER Ca2+ oscillations mediated by CaSR and revealed ER Ca2+-based functional cooperativity of CaSR. Then we described a general strategy to rationally design a new class of genetically encoded Ca2+ indicators (GECIs), with a single Ca2+-binding site and fast kinetics, by tuning rapid protein dynamics to modulate the chromophore conformational ensemble and alter the electrostatic around the chromophore. G-CatchER2 (an improved version of G-CatchER) and R-CatchER represent a general proof-of-concept.

Third, we use multiple in silico and in situ approaches including various spectroscopic and functional assays and computational modeling to provide important insights into the molecular mechanism of the receptor functions. We established a working model that several Ca2+-binding sites with endogenous ambient amino acids within the CaSR-ECD cooperatively activate the receptor. Specifically, we demonstrate the different allosteric mechanisms of TNCA and AMG-416 to modulate the receptor with the disease mutations.

These studies represent a milestone for visualizing and integrating Ca2+ dynamics, and facilitate drug discovery related to ER, CaSR, and Ca2+ dysfunction.

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