The mineralocorticoid hormone aldosterone controls sodium reabsorption and BP largely by regulating the cell-surface expression and function of the epithelial sodium channel (ENaC) in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of serum-and glucocorticoid-regulated kinase 1 (Sgk1), a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. In vivo early aldosterone-regulated mRNA now has been identified in microselected mouse distal nephron by microarray. From 22 mRNA that displayed a two-fold or more change, 13 were downregulated and nine were upregulated. Besides Sgk1, the induced mRNA include Grem2 (protein related to DAN and cerebrus [PRDC]), activating transcription factor 3, cAMP responsive element modulator, and the ubiquitin-specific protease Usp2-45. The induction of this last enzyme isoform was verified in mouse distal nephron tubule at the protein level. With the use of Hek293 cells, Xenopus oocytes, and mpkCCD c14 cells as expression systems, it was shown that Usp2-45 deubiquitylates ENaC and stimulates ENaC-mediated sodium transport, an effect that is not additive to that of Sgk1. A deubiquitylating enzyme that targets ENaC in vitro and thus may play a role in sodium transport regulation was identified within a series of new in vivo early aldosterone-regulated gene products.

␥-Aminobutyric acid, type A (GABA A ) receptors are pentameric proteins of which the majority is composed of two ␣ subunits, two ␤ subunits and one ␥ subunit. It is well documented that two different types of ␣ subunits can exist in a singles GABA A receptor complex. However, information on the abundance of such GABA A receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the ␣1, ␣2, or ␣3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABA A receptors totally insensitive to diazepam binding that contain two mutated ␣ subunits in the receptor complex, whereas receptors containing one mutated and one heterologous ␣ subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABA A receptors containing heterologous ␣ subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated ␣ subunit with those present in mouse lines containing two different mutated ␣ subunits. The data indicate that the ␣1␣1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the ␣1␣2 (13%), ␣1␣3 (15%), ␣2␣2 (12%), ␣2␣3 (2%), and ␣3␣3 combinations (4%) are considerably less expressed. Only within the ␣1-containing receptor population does the combination of equal ␣ subunits (84% ␣1␣1, 7% ␣1␣2, and 8% ␣1␣3) prevail, whereas in the ␣2-containing receptor population (46% ␣2␣2, 36% ␣2␣1, and 19% ␣2␣3) and particularly in the ␣3-containing receptor population (27% ␣3␣3, 56% ␣3␣1, and 19% ␣3␣2), the receptors with two different types of ␣ subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABA A receptors containing heterologous ␣ subunits on a brain regional level.

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