Abstract
We have recently cloned the alpha subunit of a bovine amiloride-sensitive Na+ channel (alphabENaC). This subunit shares extensive homology with both rat and human alphaENaC subunits but shows marked divergence at the C terminus beginning at amino acid 584 of the 697-residue sequence. When incorporated into planar lipid bilayers, alphabENaC almost exclusively exhibits a main transition to 39 picosiemens (pS) with very rare 13 pS step transitions to one of two subconductance states (26 and 13 pS). In contrast, the alpha subunit of the rat renal homolog of ENaC (alpharENaC) has a main transition step to 13 pS that is almost constituitively open, with a second stepwise transition of 26 to 39 pS. A deletion mutant of alphabENaC, encompassing the entire C-terminal region (R567X), converts the kinetic behavior of alphabENaC to that of alpharENaC, i. e. a transition to 13 pS followed by a second 26 pS transition to 39 pS. Chemical cross-linking of R567X restores the wild-type alphabENaC gating pattern, whereas treatment with the reducing agent dithiothreitol produced only 13 pS transitions. In contrast, an equivalent C-terminal truncation of alpharENaC (R613X) had no effect on the gating pattern of alpharENaC. These results are consistent with the hypothesis that interactions between the C termini of alphabENaC account for the different kinetic behavior of this member of the ENaC family of Na+ channels.
Highlights
A family of amiloride-sensitive Naϩ channels, the ENaCs, has recently been cloned from the colon of rats either fed a low sodium diet or treated with dexamethasone, and they have since been identified in both epithelial and non-epithelial tissues from several species [1,2,3,4,5,6,7,8]
The ␣ subunit alone can act as an amiloride-sensitive Naϩ channel [9], and other related members of the ENaC family can form a conductive pore by expression of a single cDNA (10 –12)
When membrane vesicles prepared from oocytes expressing R567X ␣bENaC were fused to planar lipid bilayers, we observed a marked difference in the gating pattern of the resultant channel as compared with that found when full-length
Summary
A family of amiloride-sensitive Naϩ channels, the ENaCs, has recently been cloned from the colon of rats either fed a low sodium diet or treated with dexamethasone, and they have since been identified in both epithelial and non-epithelial tissues from several species [1,2,3,4,5,6,7,8]. Fusion of ␣bENaC-expressing oocyte membrane vesicles to the planar lipid bilayer reveals an amiloridesensitive Naϩ channel that exhibits a distinct kinetic signature This is characterized by a main transition to 39 pS, with very rare 13 pS step transitions to one of two subconductance states (26 and 13 pS). The rat colon ␣ENaC subunit (the first cloned member of the ENaC family), exhibits a very different kinetic profile when studied under identical conditions In this case, the main transition step is to 13 pS with a second stepwise transition of 26 to 39 pS [9], and there are no long closures. We have constructed C-terminal truncated versions of both ␣ENaC subunits, expressed the respective cRNAs in Xenopus oocytes, and fused oocyte membrane vesicles to the planar lipid bilayer for electrophysiological recording
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