Cross-coupling between voltage-dependent Ca2+ channels and ryanodine receptors in developing ascidian muscle...

1. Ascidian blastomeres of muscle lineage express voltage-dependent calcium channels (VDCCs) despite isolation and cleavage arrest. Taking advantage of these large developing cells, developmental changes in functional relations between VDCC currents and intracellular Ca2+ stores were studied. 2. Inactivation of ascidian VDCCs is Ca2+ dependent, as demonstrated by two pieces of evidence: (1) a bell-shaped relationship between prepulse voltage and amplitude during the test pulse in Ca2+, but not in Ba2+, and (2) the decay kinetics of Ca2+ currents (ICa) obtained as the size of tail currents. 3. During replacement in the external solution of Ca2+ with Ba2+, the inward current appeared biphasic: it showed rapid decay followed by recovery and slow decay. This current profile was most evident in the mixed bath solution (2 % Ca2+ and 98 % Ba2+, abbreviated to '2Ca/98Ba'). 4. The biphasic profile of I2Ca/98Ba was significantly attenuated in caffeine and in ryanodine, indicating that Ca2+ release is involved in shaping the current kinetics of VDCCs. After washing out the caffeine, the biphasic pattern was reproducibly restored by depolarizing the membrane in calcium-rich solution, which is expected to refill the internal Ca2+ stores. 5. The inhibitors of endoplasmic reticulum (ER) Ca2+-ATPase (SERCAs) cyclopiazonic acid (CPA) and thapsigargin facilitated elimination of the biphasic profile with repetitive depolarization. 6. At a stage earlier than 36 h after fertilization, the biphasic profile of I2Ca/98Ba was not observed. However, caffeine induced a remarkable decrease in the amplitude of I2Ca/98Ba and this suppression was blocked by microinjection of the Ca2+ chelator BAPTA, showing the presence of caffeine-sensitive Ca2+ stores at this stage. 7. Electron microscopic observation shows that sarcoplasmic membranes (SR) arrange closer to the sarcolemma with maturation, suggesting that the formation of the ultrastructural machinery underlies development of the cross-coupling between VDCCs and Ca2+ stores.