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Open Access Publications from the University of California

Junctional cleft [Ca2+]i measurements using novel cleft-targeted Ca2+sensors

  • Author(s): Despa, S
  • Shui, B
  • Bossuyt, J
  • Lang, D
  • Kotlikoff, MI
  • Bers, DM
  • et al.

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RATIONALE:: Intracellular Ca2+concentration ([ Ca2+]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca2+release and signaling are controlled by local [ Ca2+]i in the junctional cleft ([ Ca2+]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [ Ca2+]Cleft directly are needed. OBJECTIVE:: To construct novel sensors that allow direct measurement of [ Ca2+]Cleft. METHODS AND RESULTS:: We constructed cleft-targeted [ Ca2+] sensors by fusing Ca-sensor GCaMP2.2 and a new lower Ca-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 [ Ca2+]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [ Ca2+]Cleft reaches higher levels with faster kinetics than global [ Ca2+]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca2+leak or sarcolemmal Ca2+entry may raise local [ Ca2+]Cleft above bulk cytosolic [ Ca2+]i ([ Ca2+]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [ Ca2+]Cleft-[ Ca2+]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [ Ca2+] measured without gradients as a reference point. This diastolic difference ([ Ca2+]Cleft=194 nmol/L versus [ Ca2+]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca2+leak rather than sarcolemmal Ca2+flux. CONCLUSIONS:: We have developed junctional cleft-targeted sensors to measure [ Ca2+]Cleft versus [ Ca2+]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [ Ca2+]i gradient, which could influence local Ca-dependent signaling within the junctional cleft. © 2014 American Heart Association, Inc..

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