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, 2009?and?Sun et?al., 2010). We used mostly Ratedecay for statistics because �� was often too slow to estimate in knockout mice. Compared with the wild-type, Ratedecay, but not calcium current (ICa) or ��Cm (p > 0.3), induced by depol20ms and depol20msX10 was reduced in P13�C14 CaNA��?/? mice (p?[http://en.wikipedia.org/wiki/Fluconazole Fluconazole] depol20msX10 with a pipette containing 0.05?mM BAPTA at P13�C14 rat calyces (Figures 2A�C2C). However, with 2.5?mM EGTA in the pipette, CaN457-482 significantly reduced Ratedecay without affecting the ICa or ��Cm at P13�C14 rat calyces (Figures 2D�C2F). This reduction depended on the EGTA concentration, because as the EGTA concentration increased from 0 (0.05?mM BAPTA) to 0.5, 1.25, and 2.5?mM, the Ratedecay decreased in the presence [http://www.selleckchem.com/ALK.html ALK mutation] of CaN457-482, but did not decrease in the absence of CaN457-482 (Figure?2G). The result that 2.5?mM EGTA did not affect Ratedecay in P13�C14 mice (Figure?2G, right, p?= 0.14) is consistent with a previous study (Yamashita et?al., 2010). Similar to the results shown in Figure?2D�C2G, after 20 APe at 100?Hz in P13�C14 calyces, CaN457-482 did not reduce Ratedecay in control (0.05?mM BAPTA), but significantly reduced Ratedecay in the presence of 2.5?mM EGTA (Figure?S2). We also found that a calmodulin blocker, the myosin light-chain [http://www.selleckchem.com/products/AG-014699.html Rucaparib nmr] kinase (MLCK) peptide (20?��M), did not reduce Ratedecay in pipettes containing 0.05?mM BAPTA (Figures S3A�CS3C), but reduced Ratedecay in pipettes containing 2.5?mM EGTA at P13�C14 rat calyces (Figures S3D�CS3F). These pharmacological results were consistent with the results obtained in CaNA��?/? mice. We conclude that the efficiency of CaN and calmodulin blockers depends on calcium buffer concentrations, which explains the lack of effects of CaN and calmodulin blockers in the presence of 0.5?mM EGTA (Yamashita et?al., 2010). Even in P7�C10 rat calyces where CaN blockers were effective with 0.05?mM BAPTA or 0.5?mM EGTA (Sun et?al., 2010?and?Yamashita et?al., 2010), the CaN blocker cyclosporine A inhibited endocytosis induced by 20 APe, depol20ms, and depol20msX10, but not by ten pulses of 50?ms depolarization at 10?Hz that caused a very large calcium influx (Figure?S4). Thus, the effects of CaN inhibitors depend on calcium influx, which may help resolve the CaN conflict.