Association of the epithelial sodium channel with Apx and α-spectrin in A6 renal epithelial cells

Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apr (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apr is required for the expression of amiloride-sensitive Na+ currents by cloned ENaC, Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apr. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apr, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apr is required for the functional expression of ENaC, alpha beta gamma mENaC cRNAs were coinjected into Xenopus oocytes with Apr sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apr and alpha-spectrin in A6 cells and suggest that Apr is required for the functional expression of ENaC in Xenopus epithelia.