Novel Local RYR1 Junctional Calcium Responses and Dynamics by Improved Calcium Sensor Catcher
2017
The precise spatio-temporal characteristics of Ca2+ transient are critical for the physiological regulation of Ca2+-dependent signaling processes. Because determining Ca2+ dynamics occurring in the myoplasm and sarcoplasmic reticulum (SR) under normal conditions is essential in order to fully understand changes that may occur under stressful or pathological conditions, we designed a new generation of Ca2+ sensors. Here, we report the optimization of a genetically-encoded Ca2+ sensor called “CatchER” which exhibits rapid kinetics and specific targeting to the endoplasmic reticulum (ER). We also describe the characteristics of CatchER-T’, a sensor variant with significantly improved specificities, including enhanced fluorescence at 37°C and a greater calcium dependent dynamic range. CatchER-T’ is targeted to the ER via the signal peptide of calreticulin and the ER retention sequence KDEL. Differential drug induced Ca2+ releases and ER refilling are observed for various cell types. Furthermore in electroporated mouse flexor digitorum brevis (FDB) muscle fibers, targeting CatchER-T’ close to the RyR1 channel via a sequence obtained from the junctional SR protein JP-45, revealed that local Ca2+ release via electrical stimulation is significantly greater and faster than that occurring in the SR lumen as assessed using CatchER-T’, i.e. the probe targeted to the lumen of the longitudinal SR. The impact of local ER/SR calcium dynamics in controlling cellular signaling and excitation contraction coupling will be presented.
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