Fluorescence Signal Kinetics of a New Ca2+ Probebound to Cardiac Ryanodine Receptors Point to Micro-Domain Detection of Ca2+

Abstract

To probe the micro-domains of Ca2+-signaling, we constructed a probe, calstabi-cam, targeted to cardiac ryanodine receptors (RyR2) by FKBP12.6 (calstabin 2) incorporating calmodulin and EYFP as Ca2+-sensor. 48-72h after infection of cultured rat neonatal cardiomyocytes (rNCM), global measurements of calstabi-cam fluorescence were accompanied by simultaneous recordings of Ca2+ channel (ICa) and Na+/Ca2+-exchanger (INCX) and compared to measurements of fluo-4 Ca-transients. rNCMs continued spontaneous beating after dialysis with Cs+-based internal solutions (0.1-0.2mM EGTA at −50mV) as they generated calstabi-cam fluorescence signals that coincided with INCX oscillations, like those measured by fluo-4 that were blocked by 10µM ryanodine. Activation of ICa (∼5pA/pF) triggered slowly relaxing Ca-transients of calstabi-cam fluorescence (ΔF/F0 = −0.11; n=14). Similarly, short caffeine pulses also activated long lasting calstabi-cam signals (-ΔF/F0=0.15±0.012; n=28) accompanied by INCX (1.57±0.13pA/pF; n=28). Calstabi-cam signals activated by rapid Na+ withdrawal was significantly delayed (∼230ms) compared to those triggered by ICa (∼13ms) or caffeine (∼35ms). Similar results were found in feline adult cardiac myocytes where 1µM Isoproterenol increased ICa significantly without enhancing the associated calstabi-cam transients. These results suggest that the new probe responds more effectively to Ca2+ that is released into dyadic clefts by RyR2 than by sarcolemmal Ca2+ influx via Na/Ca exchanger. The extremely slow decay kinetics of Calstabi-cam signal is consistent with the notion that [Ca2+] in the dyadic cleft may rise to much higher values than those of cytosol.