Zusammenfassung
1. Ca$^2+$ release flux across the sarcoplasmic reticulum (SR)
during cardiac excitation-contraction coupling was investigated using
a novel fluorescence method. Under whole-cell voltage-clamp conditions,
rat ventricular myocytes were dialysed with a high concentration
of EGTA (4.0 mM, 150 nM free Ca$^2+$), to minimize the residence
time of released Ca$^2+$ in the cytoplasm, and a low-affinity,
fast Ca$^2+$ indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0
mM, Kd approximately 31 microM), to optimize the detection of localized
high Ca$^2+$ in release site microdomains. Confocal microscopy
was employed to resolve intracellular Ca$^2+$ at high spatial
and temporal resolution. 2. Analytical and numerical analyses indicated
that, under conditions of high EGTA concentration, the free Ca$^2+$
change is the sum of two terms: one major term proportional to the
SR release flux/Ca$^2+$ influx, and the other reflecting the
running integral of the released Ca$^2+$. 3. Indeed, the OG-5N
transients in EGTA-containing cells consisted of a prominent spike
followed by a small pedestal. The OG-5N spike closely resembled
the first derivative (dCa$^2+$/dt) of the conventional Ca$^2+$
transient (with no EGTA), and mimicked the model-derived SR Ca$^2+$
release function reported previously. In SR Ca$^2+$-depleted
cells, the OG-5N transient also closely followed the waveform of
L-type Ca$^2+$ current (ICa). Using ICa as a known source of
Ca$^2+$ influx, SR flux can be calibrated in vivo by a linear
extrapolation of the ICa-elicited OG-5N signal. 4. The OG-5N
image signal was localized to discrete release sites at the Z-line
level of sarcomeres, indicating that the local OG-5N spike arises
from 'Ca$^2+$ spikes' at transverse (T) tubule-SR junctions (due
to the imbalance between calcium ions entering the cytosol and the
buffer molecules). 5. Both peak SR release flux and total amount
of released Ca$^2+$ exhibited a bell-shaped voltage dependence.
The temporal pattern of SR release also varied with membrane voltage:
Ca$^2+$ release was most synchronized and produced maximal peak
release flux (4.2 mM s-1) at 0 mV; in contrast, maximal total release
occurred at -20 mV (71 versus 61 microM at 0 mV), but the localized
release signals were partially asynchronous. Since the maximal conventional
Ca$^2+$ transient and contraction were elicited at 0 mV, it
appears that not only the amount of Ca$^2+$ released, but also
the synchronization among release sites affects the whole-cell Ca$^2+$
transient and the Ca$^2+$-myofilament interaction.
- 9769413
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Nutzer