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 (SR) Ca2+ release and signaling is controlled by local [Ca2+]i in the junctional cleft ([Ca2+]Cleft), the small space between sarcolemma and junctional SR. However, methods to directly measure [Ca2+]Cleft 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 (RyRs). The fluorescence pattern, affinity for RyRs and competition by un-tagged FKBP12.6 demon¬strated 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 SR 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 using GCaMP2.2-FKBP12.6 vs. GCaMP2.2, using [Ca2+] measured without gradients as a reference point. This diastolic difference ([Ca2+]Cleft=194 nmol/L vs. [Ca2+]Bulk=100 nmol/L) is dictated mainly by the SR Ca2+ leak, rather than sarcolemmal Ca2+ flux.
Conclusions: We have developed junctional cleft targeted sensors to measure [Ca2+]Cleft vs. [Ca2+]Bulk, and demonstrated dynamic differences during electrical excitation and a standing diastolic [Ca2+]i gradient which could influence local Ca2+-dependent signaling within the junctional cleft.
- junctional cleft
- local calcium signaling
- calcium signaling
- cardiac myocyte
- sarcoplasmic reticulum
- fluorescent imaging
- Received January 29, 2014.
- Revision received May 24, 2014.
- Accepted May 28, 2014.