In vivo Temperature Sensitivity of the Calcium Affinity of fluo-5F and mag-fluo4

The role of calcium ion (Ca2+) in diverse cellular signaling pathways has been identified and investigated principally by using fluorescent chelators as indicators. Using the biophysical characteristics of a fluorescent Ca2+ indicator allows investigators to convert measured fluorescence intensity into values of free Ca2+ concentration ([Ca2+]). Troublingly, the characteristics of Ca2+-indicators are dependent on numerous environmental factors. Determining the behavior of each indicator in vivo under comparable experimental conditions is necessary for quantitative measurements. Temperature is one environmental factor that strongly affects Ca2+ indicator behavior. A change in temperature can change the intrinsic physical characteristics of the indicator (e.g., Ca2+ affinity, quantum efficiency, and fluorescence lifetime), as well as change the cellular processes that affect indicator performance (e.g., extrusion by transporters, cellular pH). Therefore, experiments were designed to estimate the disassociation constants (KD) of the Ca2+ indicators fluo-5F and mag-fluo4 (loaded into the cytosol and sarcoplasmic reticulum, respectively) at room and physiological temperatures in murine ventricular myocytes. Ca2+ access to cytosolic compartment was established using a Ca2+ ionophore, while sarcoplasmic reticulum (SR) access was established by sarcolemma permeabilization with saponin in the presence of caffeine. Once access was established, buffers with known [Ca2+] were rapidly applied using a micro-perfusion system with temperature feedback control. We observed that, increasing from room temperature to 36°C caused a modest decrease in the KD of both fluo-5F and mag-fluo4. Both indicators also displayed a decrease in dynamic range when heated. Additionally, temperature-dependent extrusion was observed for indicators loaded into the cytosol, but not those loaded into the SR. These findings are important in enabling quantitative interpretation of measurements in living cells under physiological conditions.