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

Recent molecular cloning of the epithelial sodium channel (ENaC) provides t he opportunity to identify ENaC-associated proteins that function in regula ting its cell surface expression and activity. We have examined whether ENa C is associated with Apr (apical protein Xenopus) and the spectrin-based me mbrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addr essed whether Apr is required for the expression of amiloride-sensitive Na currents by cloned ENaC, Sucrose density gradient centrifugation of A6 cel l detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apr. Immunoblot analysis of proteins co-immunoprecipitating under high stri ngency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apr, and alpha-spectrin are associated in a macromolecu lar complex. To examine whether Apr is required for the functional expressi on of ENaC, alpha beta gamma mENaC cRNAs were coinjected into Xenopus oocyt es with Apr sense or antisense oligodeoxynucleotides. The two-electrode vol tage clamp technique showed there was a marked reduction in amiloride-sensi tive 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 functio nal expression of ENaC in Xenopus epithelia.