Do RYR puncta consist of one RYR or multiple RYRs? To address this question, we first estimated the number of RYRs responsible for a Ca2+ spark. Using the signal mass approach, we estimated that ICa(spark) has a peak amplitude of 2.8 pA (Fig. 1 B, c). Because under pseudophysiological conditions RYR has a unitary current of ∼0.35 pA (Mejía-Alvarez et al., 1999), 2.8 pA ICa(spark) could result from the opening of at least eight RYRs. We next examined whether a Ca2+ spark is the result of the activity of RYRs in one punctum or multiple puncta by testing whether RYRs in a punctum can activate RYRs in its closest neighboring punctum. We found that the distances between these RYRs are 0.6 ± 0.06 µm for both RYR1 and RYR2. Given that at steady-state [Ca2+], RYR1 and RYR2 have an EC50 of ∼1 µM (Fill and Copello, 2002; Laver, 2007), the probability that activation of RYRs in one punctum leads to the activation of RYRs in a neighboring punctum should be <0.5 because the maximal [Ca2+] at 600 nm from the
