Panel A shows a line graph of cytosolic calcium ion concentration over time during sequential application of thapsigargin, caffeine, and a sodium ion- and calcium ion-free solution. The x-axis represents time in seconds, and the y-axis represents cytosolic calcium ion concentration in micromolar. Panel B shows a line graph of cytosolic calcium ion concentration during sequential membrane depolarizations, with dots indicating 100-millisecond depolarizing pulses. The x-axis represents time in seconds, and the y-axis represents cytosolic calcium ion concentration in micromolar. Panel C shows a line graph of inward calcium ion currents evoked by depolarizing pulses. The x-axis represents time in milliseconds, and the y-axis represents current in picoamperes. Panel D shows a line graph of time integrals of inward calcium ion currents. The x-axis represents time in milliseconds, and the y-axis represents electrical charge in picocoulombs.
Representative experimental traces used for intracellular Ca 2+ buffer analysis. (A) Representative cytosolic Ca2+ concentration ([Ca2+]c) trace obtained from a control LVCM during sequential application of thapsigargin, caffeine, and a Na+- and Ca2+-free solution. After confirming Ca2+ transients evoked by depolarizing steps from −70 to 0 mV for 200 ms, SERCA was inhibited with thapsigargin while stimulation was continued. Caffeine (20 mM) was then applied to confirm depletion of SR Ca2+ stores. Finally, the normal Tyrode solution was replaced with 0Na+/0Ca2+ solution to inhibit NCX-mediated Ca2+ transport. (B) Representative response to a series of membrane depolarizations used to determine the relationship between Ca2+ influx and the corresponding increase in cytosolic Ca2+ concentration (). Dots indicate 100-ms depolarizing pulses. After a steady state was reached, depolarizing pulses from −40 to 0 mV were applied at 1 Hz for 6–8 repetitions to evoke inward Ca2+ currents. (C) Representative inward Ca2+ currents evoked by the depolarizing pulses shown in B. (D) Time integrals of the inward Ca2+ currents, expressed as the quantity of electrical charge and used to estimate cumulative Ca2+ entry for calculation of the Ca2+ binding ratio, as described in the Materials and methods. These traces represent an individual experiment underlying the buffer analysis presented in Fig. 6.