Junctional Cleft [Ca2+]i Measurements Using Novel Cleft-Targeted Ca2+ Sensors

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 Ca2+-sensor GCaMP2.2 and a new lower Ca2+-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+](Cleft)), 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 Ca2+-dependent signaling within the junctional cleft.