title: Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: 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.
authors.forename: Sanda
authors.initials: S
authors.lastname: Despa
authors.affiliation: 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.).
authors.forename: Bo
authors.initials: B
authors.lastname: Shui
authors.affiliation: 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.).
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.affiliation: 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.).
authors.forename: Di
authors.initials: D
authors.lastname: Lang
authors.affiliation: 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.).
authors.forename: Michael I
authors.initials: MI
authors.lastname: Kotlikoff
authors.affiliation: 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.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000235:genetics; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D056966:Excitation Contraction Coupling / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D007365:Intercellular Junctions / Q000378:metabolism; D016296:Mutagenesis; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics; D061848:Optical Imaging / Q000379:methods*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D049452:Green Fluorescent Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium
keywords: calcium signaling; myocytes, cardiac; sarcoplasmic reticulum