Paying the piper: the cost of Ca2+ pumping during the mating call of toadfish

Superfast fibres of toadfish swimbladder muscle generate a series of superfast Ca2+ transients, a necessity for high-frequency calling. How is this accomplished with a relatively low rate of Ca2+ pumping by the sarcoplasmic reticulum (SR)? We hypothesized that there may not be complete Ca2+ saturation and desaturation of the troponin Ca2+ regulatory sites with each twitch during calling. To test this, we determined the number of regulatory sites by measuring the concentration of troponin C (TNC) molecules, 33.8 mu mol per kg wet weight. We then estimated how much SR Ca2+ is released per twitch by measuring the recovery oxygen consumption in the presence of a crossbridge blocker, N-benzyl-p-toluene sulphonamide (BTS). The results agreed closely with SR release estimates obtained with a kinetic model used to analyse Ca2+ transient measurements. We found that 235 mu mol of Ca2+ per kg muscle is released with the first twitch of an 80 Hz stimulus (15 degrees C). Release per twitch declines dramatically thereafter such that by the 10th twitch release is only 48 mu mol kg(-1) (well below the concentration of TNC Ca2+ regulatory sites, 67.6 mu mol kg(-1)). The ATP usage per twitch by the myosin crossbridges remains essentially constant at similar to 25 mu mol kg(-1) throughout the stimulus period. Hence, for the first twitch, similar to 80% of the energy goes into pumping Ca2+ (which uses 1 ATP per 2 Ca2+ ions pumped), but by the 10th and subsequent twitches the proportion is similar to 50%. Even though by the 10th stimulus the Ca2+ release per twitch has dropped 5-fold, the Ca2+ remaining in the SR has declined by only similar to 18%; hence dwindling SR Ca2+ content is not responsible for the drop. Rather, inactivation of the Ca2+ release channel by myoplasmic Ca2+ likely explains this reduction. If inactivation did not occur, the SR would run out of Ca2+ well before the end of even a 40-twitch call. Hence, inactivation of the Ca2+ release channel plays a critical role in swimbladder muscle during normal in vivo function.