Figure 2. Ba²⁺ uptake by CHO cells expressing either the wild-type NCX1.1 (A) or the Δ(241–680) mutant (B), or by non-transfected CHO T cells (C).

A, after releasing internal Ca²⁺ stores with ATP + Tg (see Methods), cells expressing the wild-type NCX1.1 were treated with 1 μg ml⁻¹ gramicidin and pre-equilibrated in 100/40 Na/K-PSS. Ba²⁺ uptake was initiated by applying 2 mm Ba²⁺ in 100/40 Na/K-PSS containing 0.1 mm EGTA to chelate contaminating Ca²⁺. In trace a, 5 ml of the assay solution was superfused with continuous aspiration, while in trace b, the pre-equilibration was removed and quickly replaced with 1 ml of the assay medium. Traces represent the mean values (± s.e.m.) of 3 (trace a) or 5 (trace b) coverslips; error bars are shown for every fourth data point. B, gramicidin-treated CHO cells expressing the Δ(241–680) mutant of NCX were pre-equilibrated in 20/120 Na/K-PSS. Ba²⁺ uptake was initiated by superfusion of 5 ml of 2 mm Ba²⁺ in 20/120 Na/K-PSS (trace a, n = 4 coverslips) or by removing the pre-equilibration medium and replacing it with 1 ml of the assay medium (trace b, n = 3). For trace b, at the arrow labelled R, an additional 5 ml of 2 mm Ba²⁺ in 20/120 Na/K-PSS was applied by superfusion. For trace c, non-transfected CHO T cells were treated with gramicidin and assayed for Ba uptake under the conditions used for cells expressing the Δ(241–680) mutant. The trace represents the average of 3 experiments carried out by superfusing 5 ml of the Ba²⁺ solution and 2 experiments using the rapid replacement method. For the latter traces, an additional 5 ml of Ba²⁺ solution was superfused at the time indicated by the arrow labelled R. C, after depleting internal Ca²⁺ stores with ATP + Tg, non-transfected CHO T cells were pre-incubated for 10 min in Na-PSS + 0.3 mm EGTA without added gramicidin. Ba²⁺ uptake was measured in non-transfected CHO cells by superfusing 5 ml of 2 mm BaCl₂ in Na-PSS (trace a, n = 3) or by rapidly replacing the pre-incubation medium with 1 ml of 2 mm BaCl₂ in Na-PSS (trace b, n = 3). At the arrow labelled R, an additional 5 ml of the Ba²⁺ solution was applied by superfusion for both traces. The bars beneath the traces represent the time during which the 5 ml of solution were applied.