Both showed that the presence of exogenous A or tau in the culture medium produced a 50% decrease in cell viability, whereas co-treatments of cells with sorcin and A (molar ratio 0.2:1) or sorcin and tau (molar ratio 1:1) fully guarded cells from loss of viability. pump, and counteracts the neurotoxicity of A and tau by interacting with them. 0.001 vs. control. We have shown in previous work that hPMCA4b is the isoform most sensitive to inhibition by A [29] and tau [27]. On the other hand, Fam162a SERCA2b is usually ubiquitously expressed and is the predominant isoform in the brain [30,31]. Therefore, we examined the effects of sorcin in the presence of A and tau, on Ca2+-ATPase activity of COS membranes overexpressing the native hPMCA4b, truncated variants hPMCA4b-L1086* (L1086*) lacking the calmodulin binding domain name (CaMBD) and hPMCA4b-R1052* (R1052*), and the SERCA2b isoform. R1052* is the PMCA variant that most closely resembles SERCA, since it lacks the whole C-terminal domain. The reason for using truncated forms of PMCA was to determine whether the binding of sorcin to PMCA occurred in the C-terminal regulatory domain BMS-654457 of PMCA, which does not contain SERCA, or in other BMS-654457 domains. As shown in Physique 4, the activity of native hPMCA4b measured in the absence of sorcin was inhibited by A and tau, whereas the activity of L1086* was inhibited only by tau. In contrast, neither A nor BMS-654457 tau affected the activities of the shortest R1052* variant and SERCA2b, confirming our previous findings [25,26]. Open in a separate window Physique 4 Sorcin activates Ca2+-ATPase activities of overexpressed full hPMCA4b, their truncated variants and SERCA2b isoforms in a concentration-dependent manner and prevents their inhibition by A and tau. Twenty g of COS cells membranes overexpressing full hPMCA4b, or truncated hPMCA4b-L1086* and hPMCA4b-R1052* and SERCA2b isoforms were treated with increasing concentrations BMS-654457 of sorcin, without () or with 30 M A1-42 () or 300 nM tau (), in 25 L, and then diluted up to 1 1 mL with the assay medium plus 0.01% saponin, resulting in the indicated final concentrations of sorcin. Activities were measured as indicated in the Methods Section, after addition of 1 1 mM ATP. The 100% activities correspond to 0.226 0.02, 0.235 0.03, 0.242 0.02 and 0.190 0.003 mol. min?1. mg?1 for hPMCA4b, hPMCA-L1086*, hPMCA-L1052* and SERCA2b, respectively. Data represent mean SE of three experiments performed with three preparations. However, all Ca2+-ATPases were activated by sorcin in a concentration-dependent manner, regardless of the presence of A or tau in the incubation medium. A sorcin concentration of 25 nM completely prevented the inhibition of native PMCA by 0.75 M A or 7.5 nM tau and of the truncated L1086* by 7.5 nM tau. Above that concentration, BMS-654457 sorcin activated both native and truncated PMCAs to the same levels as those achieved in the absence of A and tau. The highest percentages of Ca2+-ATPase activation achieved with 50 nM of sorcin were as follows: 83% 5% for native hPMCA4b, 63% 3% for L1086* and 75% for R1052*. 2.3. The Ca2+-ATPase of Human Membranes Is also Modulated by Sorcin The effects of sorcin on PMCA were also investigated in membranes prepared from human undiagnosed (HC) and diagnosed AD (HAD) brains. Samples were treated, as described above, with 1 M sorcin and 30 M A or 0.3 M tau, in 25 L, and then diluted to 1 1 mL in assay medium. PMCA activity was measured as indicated in the Methods Section and the resulting values are depicted in Physique 5A. It was observed that in HC, the addition of sorcin increased PMCA activity by 1.75-fold, while A or tau, at the concentrations assayed, produced about 50% inhibition, as expected from the results of previous work. However, the activities of membranes co-treated with sorcin and A or tau were similar to those of untreated samples. These results are in agreement with those observed in purified pig brain PMCA (see Physique 1). In HAD membranes, the PMCA showed higher activity than in HC membranes, as already reported in previous studies [26,29], and it was not significantly activated by sorcin. On the other hand, A.