Mn quenching in activated zebrafish muscle fibers does not result from store-operated Ca entry

In mammalian skeletal muscle fibers, transmembrane Ca²⁺ influx is known to occur at rest and to increase in response to depolarization. In parallel to the well-identified dihydropyridine receptor (DHPR) pathway underlying this depolarization-induced Ca²⁺ influx, a tubular Ca²⁺ entry pathway activated by sarcoplasmic reticulum (SR) Ca²⁺ depletion, named store-operated Ca²⁺ entry (SOCE), has been identified. The use of the Mn²⁺ quenching technique has been instrumental for the characterization of these Ca²⁺ influxes. But, because both should be activated by depolarization, it is difficult to discriminate between these two Ca²⁺ entry pathways. In that context, the zebrafish muscle fiber is an ideal model to determine whether or not SOCE develops in response to depolarization, because the zebrafish DHPR is not conductive to any divalent cation. Using the technique of Mn²⁺ quenching of fura-2 fluorescence in voltage-clamped zebrafish fast muscle fibers, we show that depolarization pulses evoke slow transient Mn²⁺ quenching signals that persist after washout of external Mn²⁺. The Mn²⁺ quenching signal displays rate of recovery and voltage dependence correlated to the rate of recovery and voltage dependence of SR Ca²⁺ release, respectively. Our data suggest that the voltage-evoked Mn²⁺ quenching signal of zebrafish muscle fibers does not result from a Mn²⁺ influx provoked by depletion of SR Ca²⁺ content but from a displacement of Mn²⁺ accumulated on intracellular Ca²⁺ buffers by Ca²⁺ released from the SR. These findings should encourage to consider that increase in Mn²⁺ quenching can result from changes in intracellular Ca²⁺ and not from SOCE.