Identification of Murr1 as a Regulator of the Human δ Epithelial Sodium Channel

Abstract

The human ␦ epithelial sodium channel (␦ENaC) subunit is related to the ␣-, ␤-, and ␥ENaC subunits that control salt homeostasis. ␦ENaC forms an amiloride-sensitive Na ؉ channel with the ␤ and ␥ subunits. However, the in vivo function of ␦ENaC is not known. To gain insight into the function of ␦ENaC, a yeast two-hybrid screen of a human brain cDNA library was carried out using the C-and N-terminal domains of ␦ENaC. A novel ␦ENaC-interacting protein called Murr1 (mouse U2af1-rs1 region) was isolated in the C-terminal domain screen. Murr1 is a 21-kDa protein mutated in Bedlington terriers suffering from copper toxicosis. The interaction of Murr1 and ␦ENaC was confirmed by glutathione Stransferase pulldown assay and coimmunoprecipitation. To test the functional significance of the interaction, Murr1 was coexpressed with ␦␤␥ENaC in Xenopus oocytes. Murr1 inhibited amiloride-sensitive sodium current in a dose-dependent manner. In addition, deletion of the last 59 amino acids of ␦ENaC abolished the inhibition. Murr1 also bound to the ␤-and ␥ENaC subunits and inhibited ␣␤␥ENaC sodium current. Therefore, these results suggest that Murr1 is a novel regulator of ENaC.

Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

“…Since its discovery, the COMMD1 protein interaction network continues to expand (37) and includes NF-B (52, 59), epithelial sodium channel (60,61), and hypoxia-inducible factor (HIF-1␣) (22). In all of these cases, the mechanism of action of COMMD1 in facilitating protein degradation can be unified by its involvement in the ubiquitin-proteasome pathway (35)(36)(37).…”

COMMD1 (Copper Metabolism MURR1 Domain-containing Protein 1) Regulates Cullin RING Ligases by Preventing CAND1 (Cullin-associated Nedd8-dissociated Protein 1) Binding

“…The best studied of these factors is COMMD1, which participates in copper metabolism (17), NF-B-mediated transcription (18), adaptation to hypoxia (19), and electrolyte transport (20,21). A large deletion in the canine COMMD1 gene, which abolishes protein expression, leads to pathologic copper accumulation, cirrhosis, and liver failure in Bedlington terriers (17).…”

Capsazepine Is a Novel Activator of the δ Subunit of the Human Epithelial Na+ Channel

“…The reverse transcription (RT) was performed as follows. We heated the reaction mixture of total RNA (3 g) and 500 ng of an oligo(dT) [12][13][14][15][16][17][18] primer (Invitrogen) in 8 l of diethyl bicarbonate-treated water at 70°C for 10 min, chilled it for 1 min, added 11 l of the reaction buffer (as a final concentration, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, and 0.5 mM individual dNTPs), and incubated it at 50°C for 2 min. The mixture was incubated at 50°C for 90 min after the addition of 200 units of SuperScript II reverse transcriptase (Invitrogen), and then heated at 70°C for 15 min.…”

“…In addition, syntaxin 1A interacts with cell surface ENaC␣␤␥ to rapidly decrease the open probability (16). On the other hand, ENaC␦␤␥ can bind with a copper transporter, Murr1, by interacting at the C-terminal domain of ENaC␦ to decrease the activity of the channel (17). In pharmacological profiles of ENaC, it is well known that the potassium-sparing diuretics, amiloride and benzamil, inhibit the activities of ENaC␣, ENaC␦, and the complexes with ␤ and ␥ subunits (6 -11).…”