| Abstract: | A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of a Ca2+ transient. Simultaneously monitoring tension and aequorin bioluminescence provided two methods for estimating the peak myoplasmic free Ca2+] reached during the spontaneous cyclic Ca2+ release from the SR obtained in the continuous presence of a bulk solution free Ca2+] sufficiently high to overload the SR. These methods gave results in excellent agreement for the spontaneous Ca2+ release under a variety of conditions of pH and free Mg2+], and of enhancement of Ca2+ release by calmodulin. Disagreement was observed, however, when the Ca2+ transient was modified during its ascending phase. The experiments also permitted quantification of the aequorin binding within the myofibrils and determination of its operational apparent affinity constant for Ca2+ at various free Mg2+] levels. An increase of free Ca2+] at the outer surface of the SR during the ascending phase of the Ca2+ transient induced further release of Ca2+. In contrast, an increase of free Ca2+] during the descending phase of the Ca2+ transient did not cause further Ca2+ release. Varying free H+], free Mg2+], or the Na+]/K+] ratio had no significant effect on the Ca2+ transient during which the modification was applied, but it altered the subsequent Ca2+ transient. Therefore, Ca2+ appears to be the major, if not the only, ion controlling Ca2+ release from the SR rapidly enough to alter a Ca2+ transient during its course. |
