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 [ Ca 2+ ]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.
- calcium signaling
- cardiac-sarcoplasmic reticulum
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine