Figure 2. PGAM5 negatively regulates KCa3.1 activity in Jurkat T cells.

(A) Jurkat T cells over-expressing GFP-KCa3.1 (Jurkat-KCa3.1 cells) were transfected with PGAM5 or control siRNA. Lysates were IP with anti-NDPK-B or anti-GFP (KCa3.1) antibodies and IB with antibodies to 1-pHis, 3-pHis, NDPK-B and GFP antibodies as indicated. Half of the IPs were also heated at 80°C for 2 min prior to loading to ensure specificity of detection of histidine phosphorylation (histidine phosphorylation is heat-sensitive). Lysates were probed with antibodies to GFP (KCa3.1), NDPK-B, PGAM5 (to demonstrate knockdown of PGAM5), and β actin (as a loading control).

(B) KCa3.1 channel activity (TRAM-34 sensitive) was determined by whole-cell patch clamp on Jurkat-KCa3.1 T cells transfected with (i) control or (ii) PGAM5 siRNA. Shown are I/V plots of the cells, as previously described (Srivastava et al., 2006a). TRAM-34 is a specific inhibitor of KCa3.1 channel activity.

(C) Bar graph summary of TRAM-34-sensitive current in cells transfected with control or PGAM5 siRNA and PGAM5-knockdown cells rescued with siRNA-resistant PGAM5 (WT or H105A mutant), plotted at +60 mV (n = 15 to 20 cells each).

(D) Calcium flux was performed in cells re-stimulated with anti-CD3, loaded with Fura-2 AM (5 μM) and attached to a poly-L-lysine-coated coverslip for 20 min. Ca²⁺ imaging was done with an IX81 epifluorescence microscope (Olympus) and data were analyzed using OpenLab imaging software (Improvision) (n= 80–100 cells for each series), as previously described (Srivastava et al., 2009).

(E) ELISA to quantify IL-2 in the supernatants of control or PGAM5 siRNA transfected cells at 48h post transfection.

Data are representative of three independent experiments. Data are shown as mean±SEM. Statistical significance was calculated using Student’s t test; *(p<0.05); **(p<0.01); n.s. (p>0.05).

See also Figure S3.