Dissertation/Thesis Abstract

Live -cell transforms between calcium transients and FRET responses for troponin C-based calcium sensors: Their application to probe calcium in the calcium channel nanodomain
by Tay, LaiHock, Ph.D., The Johns Hopkins University, 2008, 125; 3309816
Abstract (Summary)

Ca2+ signals within nanometers of the pore of a Ca 2+ channel effect numerous Ca2+-sensitive processes. Unfortunately, direct measurement of such nanodomain Ca2+ has been difficult because these transients are beyond the optical resolution of light microscopy, and are kinetically rapid. Here, we attempt to resolve nanodomain Ca2+ at the cytoplasmic mouth of CaV2.2 (N-type) channels using a genetically encoded Ca2+ indicator (GECI) as a 'near-field' sensor. GECIs promise sustained in vivo detection of Ca2+ signals but they are sometimes challenged by inconsistent performance and often have slow kinetic responsiveness. The former challenge may arise because most sensors employ calmodulin (CaM) as the Ca2+-sensing module, such that interference via endogenous CaM may result. One class of sensors that could minimize this concern utilizes troponin C (TnC) as the Ca2+ sensor. Here, we therefore probed the reliability and kinetics of two TnC-based sensors (TN-L15 and TN-XL) within cardiac myocytes. These cells furnished substantial endogenous CaM levels and fast reproducible Ca2+ transients for testing sensor kinetics. TN-L15 and TN-XL showed highly reproducible responses but were kinetically slow relative to the rapid Ca2+ transients that accompany channel openings. Eventually, we chose to focus on TN-XL which has better kinetic performance and dynamic range than TN-L15. We therefore made CaV2.2/TN-XL fusions which showed normal electrophysiology and Ca2+ sensitivity at the surface membrane. We further tested the potential of TN-XL as a 'near-field' sensor by performing simultaneous patch-clamp electrophysiological recordings and TIRF imaging of HEK293 cells expressing CaV2.2/TN-XL channels in low intracellular buffering of 1 mM EGTA. The slow but reproducible TN-XL response observed in myocytes, together with the sensor response in 1 mM EGTA, helped define a 'forward transform' mapping Ca2+ transients to a TN-XL optical FRET readout. To probe channel nanodomain Ca2+, we recorded whole-cell currents of CaV2.2/TN-XL channels, along with the corresponding TN-XL readouts in high intracellular buffering of 10 mM EGTA that should restrict Ca2+ elevations to regions surrounding the pore of channels. The kinetics of TN-XL responses were modeled using the 'forward transform', and the analysis revealed underlying Ca2+ transients reaching more than 25-50 μM, in line with past theoretical estimates.

Indexing (document details)
Advisor: Yue, David
Commitee:
School: The Johns Hopkins University
School Location: United States -- Maryland
Source: DAI-B 69/04, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Neurosciences, Biomedical engineering
Keywords: Calcium channel, Calcium sensors, Calcium transients, Live-cell transforms, Nanodomain, Troponin
Publication Number: 3309816
ISBN: 9780549578444
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