Ca(2+)-dependent facilitation of Ca(V)2.1 and flow cytometric FRET—a quantitative, model-based approach
Lee, Shin Rong
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This dissertation addresses two distinct questions through the common lens of a quantitative, biophysical approach. Both are informed by a model-based approach to biology, and enabled by cutting-edge experimental systems that permit the perturbation and simultaneous measurement of cellular signals. The first part of this thesis describes how the brain-predominant voltage-gated Ca2+ channel (CaV2.1) is regulated by its permeant ion Ca2+. By pairing novel electrophysiological techniques with Ca2+ imaging and uncaging, intracellular Ca2+ can be directly controlled through light, and the Ca2+-dependent effects on channel gating resolved independently from confounding processes like ongoing voltage activation of channels. With this unprecedented control, we find surprisingly that Ca2+-dependent facilitation of CaV2.1 is larger, faster and more Ca2+-sensitive than previously imagined. These properties suggest that CaV2.1 furnishes exceptionally strong activity-dependent enhancement of Ca2+ entry throughout the nervous system, with important repercussions for downstream Ca2+-dependent processes like neuronal plasticity. The second part of this thesis moves to consider a high-throughput method for illuminating protein structure and function through fluorescence. It describes the development of a technique to measure with a flow cytometer single-cell protein levels and Förster resonance energy transfer (FRET) efficiencies in single live cells. With this flow cytometric FRET, we show that binding curves can be rapidly constructed, and binding affinities obtained comparable to those using isothermal calorimetry. We applied this technique, with the aid of a FRET-based PKA activity sensor, to uncover the biochemical ramifications of a recently discovered Cushing’s disease-causing mutant (L206R) of protein kinase A (PKA). We find that this point mutation within the catalytic domain of PKA not only differentially disrupted its ability to bind to select partners, but also caused an underlying catalytic deficiency. Our results provide mechanistic insight into the pathogenesis of L206R-mediated Cushing’s syndrome, guiding potential future therapeutic strategies. They also showcase flow cytometric FRET as a rapid and convenient tool for assaying protein biochemistry within the highly relevant context of living cells.