Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

Abstract

Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the ␣, ␤, and ␥ subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both ␣-and ␤-but not ␥-ENaC. Western blots showed that both ␣-and ␤-but not ␥-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against ␣-ENaC but not by antisense oligonucleotide against ␤-ENaC, indicating that only the ␣ subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 M) reduced the frequency of observance of 21-pS ␣-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from ␣-and ␤-ENaC. After expression of exogenous ␥-ENaC, aldosterone again reduced the frequency of observance of the 21-pS ␣-ENaC channel but induced the appearance of a 5-pS channel, presumably a ␣␤␥-ENaC channel. In the absence of aldosterone, the ␣ subunit forms an ␣-cryptic channel that is activated by stretch, and in the presence of aldosterone, ␤ and ␣ subunits together form an active channel that is modulated by aldosterone.The superfamily of proteins to which the epithelial sodium channel (ENaC) 1 belongs generally mediates cation transport across cell membranes. ENaC, itself, is usually associated with sodium transport across the apical membrane of a variety of epithelia including the colon, lung, and kidney. Since 1994, when ENaC was initially cloned from rat colon (1), the biophysical properties and molecular structure of ENaC have been studied extensively. Several lines of evidence suggest that ENaC, including human ENaC (hENaC), is typically composed of three subunits, ␣, ␤, and ␥, and that all three subunits are required to form a functional ␣␤␥-ENaC channel complex (1-6). In heterologous expression systems, maximal expression of the hENaC channel requires co-expression of all three subunits (7, 8), and in oocytes, expression of ␣-ENaC cRNA alone produces little expression of any amiloride-sensitive currents. However, in other cell types, expression of the exogenous ␣-ENaC subunit alone can form a stretch-activated nonselective cation channel (9), and a similar nonselective cation channel has also been described in native lung epithelial alveolar type II cells (10 -12). This lung cation channel appears to be formed from ␣-ENaC alone and is equally permeable to Na ϩ and K ϩ , is sensitive to steroid hormones, and has a h...

Regulation of Epithelial Sodium Channel Trafficking by Ubiquitination

“…These are domains that contain high concentrations of inositol phospholipids and the enzymes that produce the lipids (25)(26)(27). Some fraction of ENaC is present in lipid rafts (28,29) that contain high levels of inositol phospholipids and other charged lipids (30)(31)(32)(33). Artificial disruption of lipid rafts reduces ENaC activity, but the prevalence of lipid rafts also depends upon hormonal factors (e.g., tumor necrosis factor-a [30], activators of protein kinase C, or PTEN tumor suppressor).…”

“…The reported values for the half-life of ENaC in the plasma membrane vary widely, from as short as 15 minutes (5) to over 24 hours (32). This large variability almost surely has to do with exactly what form of ENaC is being measured and in which cellular system.…”

“…G protein receptor kinase 2 (GRK2) also phosphorylates Nedd4-2 to reduce interaction with ENaC and prevent ubiquitination and degradation and, finally, Nedd4-2 is phosphorylated and inhibited by cAMP-dependent protein kinase A. b to form a functional channel; and finally, (3) the stability of membrane surface ENaC. Another reason it is difficult to correlate the biochemical data to the functional stability of ENaC is that there are considerable data suggesting that changes in a-, b-, and g-ENaC subunit levels in the cell surface are not coordinately regulated (21,31,32) meaning that the half-life of each subunit may differ from one another. Therefore, it is difficult to accurately correlate the quantitative changes in the expression levels of ENaC (determined using biochemical approaches) to the actual functional stability of ENaC.…”

“…In attempts to estimate the physiological contribution of the channels and to overcome the technical limitations of biochemical assays, a few groups have measured the decrease of amiloride-sensitive whole cell current in oocytes and MDCK cells (20,26) under experimental conditions that prevent newly synthesized channels from being delivered to the plasma membrane. Since post-translational modifications and trafficking of ENaC in heterologous expression systems sometimes have characteristics that differ from channels found endogenously in native epithelial cells (10,32), the stability of ENaC in cells overexpressing ENaC may differ significantly from the sodium channels found in native cells. Furthermore, amiloride may inhibit transporters and channels other than ENaC (8,9), and therefore, the half-life of ENaC observed based solely on the decline of amiloride-sensitive current may not actually reflect the half-life of individual ENaC.…”

Human Taste: Peripheral Anatomy, TasteTransduction, and Coding

“…The molecular basis for this salt taste aspect is unclear but the heat-and capsaicinsensitive TRP receptor VR1 has been implicated [137]. In addition, hormones like aldosterone could change the expression levels of these channels, thus regulating the sensitivity to salty stimuli and to suppression of these inhibitors [138]. At present, the transduction mechanism for human salt taste is unknown.…”