FIGURE 6.

Tandem affinity purification of human α-, β-, and γ-ENaC from MDCK stable cell lines and channel function of α-ENaC reconstituted in lipid bilayer. A, purified human α-, β-, and γ-ENaC channel proteins visualized by Coomassie Blue staining (left panel) and WB (right panel), as indicated by the arrow. All proteins were purified from MDCK cell lines stably transfected with α-, β-, and γ-ENaC cDNAs in the pGTAP3F vector. Antibodies against α-, β-, and γ-ENaC (Santa Cruz Biotechnology) were used for WB. B, representative tracings obtained using purified α-ENaC channels reconstituted in the lipid bilayer system (left panel) and current-voltage relationship (right panel). Single channel activities were recorded under an asymmetrical condition (150/15 mm NaCl on cis/trans, i.e. intra/extracellular, compartments). Dotted lines indicate closed states.