DIRECT MEASUREMENT OF SR RELEASE FLUX BY TRACKING CA2+ SPIKES IN RAT CARDIAC MYOCYTES

1. Ca2+ release flux across the sarcoplasmic reticulum (SR) during car diac excitation-contraction coupling was investigated using a novel fl uorescence method. Under whole-cell voltage-clamp conditions, rat vent ricular myocytes were dialysed with a high concentration of EGTA (4.0 mM, 150 nM free Ca2+), to minimize the residence time of released Ca2 in the cytoplasm, and a low-affinity, fast Ca2+ indicator, Oregon Gre en 488 BAPTA-5N (OG-5N; 1.0 mM, K-d approximate to 31 mu M), to optimi ze the detection of localized high [Ca2+] in release site microdomains . Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution. 2. Analytical and numerical anal yses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportiona l to the SR release flux/Ca2+ influx, and the other reflecting the run ning integral of the released Ca2+. 3. Indeed, the OG-5N transients in EGTA-containing cells consisted of a prominent spike followed by a sm all pedestal. The OG-5N spike closely resembled the first derivative ( d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mim icked the model-derived SR Ca2+ release function reported previously. In SR Ca2+-depleted cells, the OG-5N transient also closely followed t he waveform of L-type Ca2+ current (I-Ca). Using I-Ca as a known sourc e of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrap olation of the I-Ca-elicited OG-5N signal. 4 The OG-5N image signal wa s localized to discrete release sites at the Z-line level of sarcomere s, indicating that the local OG-5N spike arises from 'Ca2+ spikes' at transverse (T) tubule-SR junctions (due to the imbalance between calci um ions entering the cytosol and the buffer molecules). 5. Both peak S R release flux and total amount of released Ca2+ exhibited a bell-shap ed voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mM s(-1)) at 0 mV; in contrast, maxima l total release occurred at -20 mV(71 versus 61 mu M at 0 mV), but the localized release signals were partially asynchronous. Since the maxi mal conventional [Ca2+] transient and contraction were elicited at 0 m V, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole-cell Ca2+ transi ent and the Ca2+-myofilament interaction.