Sanda Despa1, Bo Shui2, Julie Bossuyt2, Di Lang2, Michael I Kotlikoff2, Donald M Bers1. 1. From 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. 2. From 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.).
Abstract
RATIONALE: Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed. OBJECTIVE: To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. METHODS AND RESULTS: We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L versus [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca(2+) leak rather than sarcolemmal Ca(2+) flux. CONCLUSIONS: We have developed junctional cleft-targeted sensors to measure [Ca(2+)]Cleft versus [Ca(2+)]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca(2+)]i gradient, which could influence local Ca(2+)-dependent signaling within the junctional cleft.
RATIONALE: Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed. OBJECTIVE: To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. METHODS AND RESULTS: We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulumCa(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L versus [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca(2+) leak rather than sarcolemmal Ca(2+) flux. CONCLUSIONS: We have developed junctional cleft-targeted sensors to measure [Ca(2+)]Cleft versus [Ca(2+)]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca(2+)]i gradient, which could influence local Ca(2+)-dependent signaling within the junctional cleft.
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