Abstract
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 Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx 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 Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx 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 Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.
Highlights
Epithelial Naϩ channels mediate entry of Naϩ from the luminal fluid into the cells during the first stage of electrogenic transepithelial Naϩ transport across Naϩ-reabsorbing epithelia [1, 2]
The relationship of the cloned ENaC to the biochemically purified Naϩ channel has been a point of contention, Rokaw et al [11] have recently presented data indicating that ␣, , and ␥ ENaC are components of the epithelial Naϩ channel biochemically isolated from A6 cells
Naϩ-reabsorbing epithelial cells are polarized with their plasma membranes divided into two structurally and biochemically distinct domains: the apical, which faces the luminal compartment, and the basolateral, which rests on the basement membrane and is in contact with the interstitial compartment
Summary
Vol 274, No 33, Issue of August 13, pp. 23286 –23295, 1999 Printed in U.S.A. Association of the Epithelial Sodium Channel with Apx and ␣-Spectrin in A6 Renal Epithelial Cells*. 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 Apx and ␣-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia. The molecular cloning of the highly Naϩ-selective epithelial sodium channel (ENaC) from a variety of epithelial cells, including A6 cells, has revealed that is composed of three homologous subunits, ␣, , and ␥ [2, 9, 10] This channel has a single channel conductance of 4 pS when expressed in Xenopus oocytes and exhibits ion selectivity, gating kinetics, and an amiloride-pharmacological profile similar to that of the 4 pS, highly Naϩ-selective channel expressed in native Naϩ-reabsorbing epithelia [2, 9, 10]. Kleyman and co-workers [12] have revealed that the monoclonal antibody RA 6.3, which was used to biochemically isolate a Naϩ channel complex from A6 cells, recognizes the amiloride-binding site on ␣ ENaC
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