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+]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 Ca2+-dependent signaling within the junctional cleft.