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.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 69/04, Dissertation Abstracts International|
|Subjects:||Neurosciences, Biomedical engineering|
|Keywords:||Calcium channel, Calcium sensors, Calcium transients, Live-cell transforms, Nanodomain, Troponin|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be