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Published on: May 19, 2017
Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors
Sanda Despa1, Bo Shui2, Julie Bossuyt2
1From the Department of Pharmacology, University of California, Davis (S.D., J.B., D.L., D.M.B.); Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington (S.D.); and Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY (B.S., M.I.K.). dmbers@ucdavis.edu s.despa@uky.edu.
Researchers developed new sensors to measure calcium in heart cell junctions. These sensors reveal higher, faster calcium levels in the junctional cleft during heartbeats, potentially explaining arrhythmias.
Area of Science:
- Cardiovascular Physiology
- Molecular Biology
- Biophysics
Background:
- Intracellular calcium concentration ([Ca(2+)]i) acts as a versatile second messenger, with signaling varying across subcellular microdomains.
- In cardiac cells, local [Ca(2+)]i within the junctional cleft ([Ca(2+)]Cleft) regulates sarcoplasmic reticulum Ca(2+) release but direct measurement methods are lacking.
Purpose of the Study:
- To engineer novel calcium sensors for direct measurement of [Ca(2+)]Cleft.
- To investigate the dynamic changes and spatial gradients of [Ca(2+)]Cleft during cardiac excitation-contraction coupling.
Main Methods:
- Constructed cleft-targeted calcium sensors by fusing GCaMP2.2 variants with FKBP12.6, a ryanodine receptor-binding protein.
- Validated sensor localization and function in adult rat myocytes using fluorescence and competition assays.
- Measured [Ca(2+)]Cleft and global cytosolic [Ca(2+)]i ([Ca(2+)]Bulk) dynamics during cellular excitation.
Main Results:
- FKBP12.6-tagged sensors successfully localized to the junctional cleft, enabling direct [Ca(2+)]Cleft measurement.
- [Ca(2+)]Cleft exhibited higher peak levels and faster kinetics compared to [Ca(2+)]Bulk during excitation-contraction coupling.
- A standing diastolic gradient was observed ([Ca(2+)]Cleft = 194 nmol/L vs. [Ca(2+)]Bulk = 100 nmol/L), primarily driven by sarcoplasmic reticulum calcium leak.
Conclusions:
- Developed novel junctional cleft-targeted sensors for simultaneous measurement of [Ca(2+)]Cleft and [Ca(2+)]Bulk.
- Demonstrated significant dynamic differences in [Ca(2+)]Cleft during electrical excitation.
- Identified a diastolic [Ca(2+)]i gradient in the junctional cleft, potentially influencing local calcium-dependent signaling and contributing to arrhythmias.
