News Physiol Sci 15: 161-165, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 4, 161-165,
August 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
Toward Understanding the Role of Methylation in Aldosterone-Sensitive Na+ Transport
James D. Stockand,
Robert S. Edinger,
Douglas C. Eaton and
John P. Johnson
J. D. Stockand is in the Department of Physiology, University of Texas Health Science Center San Antonio, San Antonio, Texas 78284-7756. D. C. Eaton is at the Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322; R. S. Edinger and J. P. Johnson are in the Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
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Abstract
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Proper endocrine regulation of Na+ reabsorption by renal principal cells is the primary means in mammals for maintaining blood pressure. Aldosterone increases Na+ reabsorption by activating luminal Na+ channels; however, the signal transduction pathway of aldosterone is not fully understood. Cellular methylation is necessary for aldosterone signaling to the luminal Na+ channel. We describe the enzymes, regulators, and effectors of aldosterone-mediated methylation relevant to Na+ reabsorption.
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Introduction
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Terrestrial vertebrates, including humans, maintain chronic blood pressure by tight regulation of Na+ and water reabsorption at the distal tubule of the kidney. The principal cell in the cortical collecting duct (CCD) is responsible for regulated Na+ reabsorption. Aldosterone is the most important systemic hormone controlling discretionary Na+ reabsorption at the CCD.
Figure 1
shows the idealized cell model of a Na+ reabsorbing epithelial cell, such as the principal cell. The rate-limiting step in electrogenic Na+ reabsorption is entry across the luminal plasma membrane. Entry of Na+ is down its electrochemical gradient through a Na+-selective ion channel. This channel has been cloned and named the epithelial Na+ channel (ENaC). ENaC in principal cells is well characterized (for recent reviews, see Refs. 4 and 5). Abnormal ENaC activity resulting from channel mutation or dysfunctional hormone signaling results in improper regulation of blood pressure due to plasma fluid volume imbalances. Aldosterone modulates the activity of ENaC to regulate the rate of discretionary Na+ reabsorption. Serosal Na+-K+-ATPases maintain the electrochemical gradient at the expense of chemical energy (ATP catabolism). Thus the active transport of Na+ across the serosal membrane allows for the restrictive diffusion across the luminal membrane, with the activity of the luminal entry pathway being rate limiting and modulated in response to aldosterone.
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FIGURE 1. A model Na+-reabsorbing epithelial cell. The cell entry of Na+ through the luminal epithelial Na+ channel (ENaC) is rate limiting for Na+ reabsorption. Aldosterone modulates the activity of ENaC through the actions of aldosterone-induced proteins.
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Aldosterone has a biphasic action with respect to increased Na+ reabsorption: an early phase (4–6 h) that results from transcription/translation of unknown signaling molecules and a later phase (12–48 h) that results from an elevation in channel and pump expression (5). Both phases have an absolute requirement for expression of aldosterone-induced proteins (AIPs). However, the AIPs have only been partially characterized. Although the early action of aldosterone has a role in dynamic regulation of blood pressure in response to hourly changes in plasma Na+ balance, the more trophic actions of the steroid set the chronic capacity for Na+ reabsorption at the CCD. How the early and late phases of aldosterone action are related is currently unclear.
The laboratory of Johnson was the first to show that aldosterone increases methylation of both lipids and proteins in epithelia (reviewed in Ref. 10). The time course of this methylation (4 h) correlated well with the early actions of aldosterone. Moreover, methylation-promoting agents, such as S-adenosyl-L-methionine (SAMe, also abbreviated SAM and AdoMet), increased Na+ entry across the luminal membrane. That the magnitude of increase in Na+ transport was similar with aldosterone and SAMe and that aldosterone and SAMe were not additive suggests that both work through the same signaling pathway. Several other investigators have also now shown that aldosterone increases substrate methylation in epithelia (9, 10, 12). Also confirmed was that SAMe increases Na+ channel activity (4, 6, 9). Several investigators have also reported that a pharmacological inhibitor of methylation attenuates aldosterone-induced Na+ reabsorption, showing further a dependency of transport on methylation (4, 10, 11).
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Biological methylation
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Diverse physiological molecules are methylated, including nucleic acids, lipids, proteins, and hormones. An idealized protein methylation reaction is depicted in Fig. 2. Certain types of protein methylation are analogous to phosphorylation, with both being reversible molecular switches that control protein activity/locale in physiological milieus and time courses. Protein methyltransferases use SAMe as a donor to transfer a methyl moiety to a nucleophilic oxygen, nitrogen, or sulfur in a polypeptide chain to form methyl esters, amines, and amides, respectively (recently reviewed in Ref. 3). Protein methyltransferases are classified into two major groups: those that modify carboxyl groups to form methyl esters and those that modify sulfur and nitrogen. Since methylation catalyzed by the prior enzymes is reversible and known to regulate protein activity, O-carboxymethylation likely plays a role in dynamic cellular signal transduction. In contrast, methylation catalyzed by the latter enzymes is irreversible and thus does not have a role in dynamic signaling. This latter sort of methylation expands the number of physiological amino acids, possibly resulting in static changes in protein function, oligomerization, and localization.
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FIGURE 2. An idealized protein methylation reaction. Pr, protein; SAH, S-adenosyl-L-homocysteine; SAMe, S-adenosyl-L-methionine.
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Four types of protein carboxymethyltransferases are well established. Type I modifies glutamate residues contained in bacterial chemoreceptors, type II modifies isoaspartate/aspartate residues that likely target proteins for repair or degradation, type III modifies carboxyisoprenylcysteine (protein carboxyisoprenylcysteine methyl transferase; PCMT), and type IV modifies carboxyleucine (reviewed in Ref. 3). In addition to these carboxymethyltransferases, protein methyltransferases that N-methylate amino acids, such as arginine (protein arginine methyl transferase; PRMT), have also been described.
The best-described methylation involved in signal transduction in eukaryotes is methyl esterification of proteins terminating with carboxyisoprenylcysteines. Small, monomeric G proteins, such as p21ras, nuclear lamin B, cyclic nucleotide phosphodiesterase, and subunits of trimeric G proteins all contain such carboxy-terminal cysteine isoprenoids (3). Methylation of these signaling molecules controls their activity or cellular localization, with Ras being specifically localized to the inner leaflet of the plasma membrane upon methyl esterification. Reversible methylation of other signaling proteins, such as phosphoprotein phosphatase 2A (PP2A), has also been reported (3). For PP2A, methylation of a carboxyleucine is catalyzed by an enzyme different from that leading to modification of carboxyisoprenylcysteine.
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Aldosterone-induced methyltransferase is necessary for Na+ reabsorption
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Inhibition of mineralocorticoid-induced Na+ transport by competitive inhibitors of isoprenylcysteine-O-carboxymethyltransferase [N-acetyl-S-farnesyl-L-cysteine (AFC) and farnesylthiosalicylic acid] show that substrate methyl esterification by PCMT is necessary for aldosterone-mediated Na+ reabsorption (1, 12). A recent report by Blazer-Yost and colleagues (2) showing that N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), a competitive inhibitor of all known methyltransferases that modify carboxy-terminal isoprenylcysteines, abolishes aldosterone-induced transport is also consistent with the hypothesis that PCMT is necessary for aldosterone signaling to ENaC.
Aldosterone induces both a GTP-dependent increase in formation of peptide methyl esters in vitro and a GTP-dependent methylation of a 95-kDa protein substrate in vivo (6, 9, 10, 12). PCMT is the only protein methyltransferase well documented to be stimulated by GTP.
The recent cloning of the gene coding PCMT and the production of a specific antibody against PCMT allowed for the direct investigation of the hypothesis that this transferase is involved in mediating aldosterone-induced Na+ transport. Aldosterone induces PCMT activity without affecting enzyme number, suggesting that this methyltransferase is not an aldosterone-induced protein but that it ultimately is regulated by an AIP (12). Aldosterone-induced activation of PCMT results in increased protein methylation in vivo. Overexpression of PCMT potentiates aldosterone-induced Na+ transport but does not mimic all steroid action, suggesting that PCMT activity is not rate limiting for Na+ transport in the absence of steroid but that it can become limiting in the presence of steroid. This observation is interesting because it suggests that either a regulator or effector of PCMT, or a permissive parallel signaling pathway, is limiting in the absence of steroid.
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Substrates of aldosterone-stimulated methyltransferase relevant to Na+ reabsorption
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Although diverse molecules can be methylated in physiological systems, all current biochemical and electrophysiological results support the idea that methylation of protein is critical for induction of aldosterone-sensitive Na+ transport (10). Two candidate protein substrates for aldosterone-increased methylation have emerged: the ß-subunit of ENaC and the small, monomeric GTP binding protein p21ras.
The initial suggestion that ßENaC might be a methylation substrate were findings showing that a 90- to 95-kDa apical membrane protein (possibly part of the luminal Na+ channel) was methylated in response to steroid treatment (6, 10). Glycosylated ßENaC is ~90–95 kDa. We have shown that aldosterone stimulates methylation of ßENaC in membranes of renal epithelial cells in a GTP-dependent manner (9). Also consistent with ßENaC being a substrate for steroid-stimulated methylation are in vivo results showing that aldosterone induces methylation of proteins ranging from 85 to 95 kDa (1). Moreover, in vitro translated ßENaC, but not 
- or 
ENaC, proved to be a substrate for methylation by enzymes contained in a membrane preparation from renal epithelial cells (9). Further evidence that supports ßENaC as a methylation substrate are electrophysiological results documenting activation of ENaC in excised patches (4) and reconstituted in planar lipid bilayers (6, 9) by addition of SAMe to the intracellular face of the ion channel. Both of these electrophysiological maneuvers disrupt cellular signaling pathways by isolating integral and peripheral plasma membrane proteins from other cellular proteins, leaving only the ion channel and closely associated proteins available for methylation. If ßENaC is indeed the substrate (or one of the substrates) of aldosterone-stimulated methylation, these electrophysiological studies suggest the site to be on the intracellular portion of the molecule. Interestingly, there is no clear O-carboxymethylation consensus site in either the carboxy or amino terminal, putative cytosolic domains of ßENaC. However, the carboxy terminus of ßENaC contains arginine residues flanked by a proline-rich region. Arginines in such a motif can be N-methylated by PRMT. At present, though, it is unclear which amino acid(s) in ßENaC is targeted for methylation.
Another possible substrate for aldosterone-induced methylation is p21ras. In renal epithelia, methyl esterification of Ras was recently shown to be elevated ~14-fold in response to aldosterone (1). Methyl esterification is required for proper membrane localization of Ras. Other recent results show that Ras translocates to the plasma membrane within 4 h in response to steroid treatment (13). Further support for the idea that aldosterone-sensitive Ras methylation is important for Na+ transport are two observations: 1) PCMT, which methylates carboxy-terminal CAAX (cysteine, aliphatic residue, any residue) consensus sites (such as that of Ras), is activated by steroid and necessary for transport, and 2) the Ras protein is necessary for Na+ transport and ENaC activity in renal epithelia (1, 12, 13).
Interestingly, K-Ras is an aldosterone-induced protein necessary for aldosterone-stimulated Na+ reabsorption (7, 13). Downstream effectors of Ras signaling are also stimulated by aldosterone in a Ras-dependent manner. The ultimate effect of aldosterone-activated K-Ras is to sustain activity of the luminal Na+ channel. Overexpression of K-Ras increases basal Na+ reabsorption in the absence of steroid, suggesting that the levels of K-Ras are limiting for regulated transport in the absence of aldosterone. Recall that overexpression of PCMT failed to affect Na+ transport in the absence of steroid but did potentiate aldosterone action, suggesting that PCMT is not limiting in the absence of steroid but becomes limiting in its presence. These two observations together suggest that at rest the levels of K-Ras are limiting and that subsequent to aldosterone-increased K-Ras levels the aldosterone-sensitive PCMT activity becomes limiting. Thus activation of PCMT by aldosterone converges with steroid induction of Ras synthesis. This methylated Ras, localized to the apical membrane, is then able to affect ENaC. The mechanism by which methylated Ras regulates ENaC remains to be elucidated.
Another candidate protein substrate for aldosterone-relevant methylation is PP2A. Although no direct evidence supports the notion that PP2A is methylated in response to aldosterone, this protein is a known substrate for methylation and may be important to the proper regulation of ENaC.
Figure 3 shows that the aldosterone-induced activity of ENaC in cell-attached patches created on renal epithelial cells with decreased methyltransferase activity is markedly lower than the activity in cells with normal transferase activity. Moreover, the activity in transferase-decreased cells treated with steroid is similar to that in normal cells not treated with steroid. Thus these and other direct single-channel experiments (4, 6, 9) demonstrate that the final effector of aldosterone-relevant methylation is the apical Na+ channel. What remains unclear are the effectors intermediate to methyltransferase and ENaC.
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FIGURE 3. ENaC activity is regulated by methylation. ENaC current traces in cell-attached patches were made on renal epithelial cells treated with aldosterone that had normal methyltransferase activity (control) and those that had methyltransferase activity depressed (reduced methyltransferase activity). These cells were bathed in physiological saline, and no pipette potential was applied. Inward current is down, with arrows indicating the closed state.
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For ßENaC methylation, it is likely that no intermediate signaling protein exists. Moreover, it is possible that subunit methylation either directly modulates channel gating to control open probability or increases ENaC activity by modifying channel assembly or insertion/retrieval. However, the role of putative accessory proteins that recognize the methylated subunit are unknown.
It is also possible that methylated Ras directly regulates ENaC gating or insertion/retrieval. Alternatively, Ras could signal to ENaC through a number of different effectors. Phosphatidylinositol 3-kinase (PI3-kinase) is one of only a few well-documented first effectors of Ras. Interestingly, PI3-kinase modulates ENaC activity (8). Aldosterone, via Ras signaling, also activates the kinase cascade associated with cell proliferation and differentiation (13). It is possible that any of these kinases and modified gene expression in response to activation of the kinase cascade could regulate ENaC activity. However, this latter possibility is not likely, since ENaC can be activated by methylation when the channel is physically separated from the nucleus.
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Mechanisms of aldosterone induction of methyltransferase
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The cellular mechanism of aldosterone-increased methylation remains unclear. Support for both post-translational and metabolic regulation of methyltransferase activity in response to aldosterone has been provided (11, 12). Several investigators have documented a steroid-induced increase in in vitro substrate methylation in whole cell lysate and extracts enriched in luminal membrane (9, 10, 12). This aldosterone-mediated increase in activity in a cell-free system, in conjunction with the observation that methyltransferase levels do not increase in response to steroid (12), suggest some form of post-translational modification of a relevant transferase.
The predominate intracellular methyl-donating molecule, SAMe, is metabolized to S-adenosyl-L-homocysteine (SAH) during substrate methylation. This endproduct is a potent feedback inhibitor of all methylation reactions. The only enzyme in vertebrates capable of SAH hydrolysis is S-adenosyl-L-homocysteine hydrolase (SAHHase). Thus regulation of cellular SAH levels by SAHHase may be an important site for controlling methylation and possibly Na+ transport. Several laboratories have reported that inhibition of SAHHase activity decreases aldosterone-induced Na+ influx, current, and Na+ channel activation (4, 10). Recently, we described the cellular mechanisms of regulation of Na+ transport by SAHHase (11). Aldosterone induces SAHHase activity within 4 h. This increase in activity results in increased SAH hydrolysis, which leads to a concomitant increase in substrate methylation and Na+ reabsorption. Thus aldosterone induction of SAHHase activity is a form of metabolic regulation of the methylation reaction relevant to Na+ transport.
In summary, aldosterone stimulates a methylation reaction that is necessary for the early phase of regulated Na+ reabsorption (Fig. 4). The ultimate effector of steroid-induced methylation is the luminal Na+ channel. However, a number of intriguing questions remain concerning this signal transduction pathway. Although aldosterone stimulates diverse methyltransferase activities (leading to increased phospholipid and protein methylation), the mechanism of steroid action is indirect with no apparent change in transferase protein levels. The pathways involved in regulation of transferase activity remain to be elucidated but likely involve modulation of SAHHase activity and post-translational modification of the various methyltransferases. Although PCMT is critical to Na+ reabsorption, the roles for other methyltransferases remain to be investigated. Thus the specific transferases relevant to Na+ reabsorption and the actions of aldosterone on these proteins need to be further defined. Both K-Ras, an aldosterone-induced protein, and ßENaC are methylated in response to aldosterone. The mechanism of ENaC activation by these methylated proteins has not been investigated. Moreover, a role for other methylation substrates (like PP2A) in aldosterone signaling needs to be pursued further, and effectors intermediate from the methylation substrate to ENaC need to be elucidated. The actions of aldosterone on epithelia are known to be pleotropic, with early and late phases. Methylation reactions may be involved in more than one phase of aldosterone signal transduction.
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FIGURE 4. A model of aldosterone signaling to ENaC. Aldosterone-stimulated methyltransferase methylates K-Ras (also an aldosterone-induced protein) and the ß-subunit of ENaC. These methylated proteins subsequently lead to increased ENaC activity, though the mechanism of this action remains to be described. One methyltransferase likely to be stimulated by aldosterone is protein carboxyisoprenylcysteine methyl transferase (PCMT).
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References
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Introduction
Biological methylation
