Figure 3.

Increased levels of CRT increase Ca²⁺ uptake capacity in vitro. ER-enriched membrane vesicles (fraction 2, Fig. 1), were obtained from CRT transgenic, Nt GFP:4 transgenic, and wild-type cell lines as indicated in “Materials and Methods.” ATP-dependent ⁴⁵Ca²⁺ uptake was performed on the ER-enriched vesicles (see “Materials and Methods”). The ATP-dependent Ca²⁺ uptake was measured in presence and absence of 3 mm ATP, and is shown as Δ ATP. Black symbols denote heat-shocked cells and white symbols denote non-heat-shocked cells. A, ER-enriched membrane vesicles from CRT overproducing (triangles), wild-type (squares), and CRT antisense (circle) cell lines were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹ see “Materials and Methods”). The ⁴⁵Ca²⁺ recovered was 11.1 ± 0.7 nmol ⁴⁵Ca²⁺ mg protein⁻¹ at 20 min for heat-shocked wild type. B, ER-enriched membrane vesicles from GFP expressing (triangles) and wild-type (squares) cell lines were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹). Data (mean of two values ± the range) are shown from one experiment. The experiment has been repeated at least three times with consistent results. The increase in ⁴⁵Ca²⁺ uptake in the CRT sense lines compared with heat-shocked wild type was 2.0 ± 0.3-fold (mean ± sd) for three experiments.