Abstract-Na v 1.5, the cardiac isoform of the voltage-gated Na ϩ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na v 1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na v 1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na v 1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na v 1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na v 1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na v 1.5 and Nedd4-2. Na v 1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na v 1.5. Consistent with the functional findings, localization at the cell periphery of Na v 1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na v 1.5, suggesting that Nedd4-2 is a specific regulator of Na v 1.5. These results demonstrate that Na v 1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na v 1.5 by decreasing the channel density at the cell surface.

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.

The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na(v)1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na(v)1.2, Na(v)1.3, and Na(v)1.5 indicated that mouse brain Nedd4-2 binds to the Na(v) PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na(v)1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K(d) of approximately 55 muM. We tested the binding properties and the ability to ubiquitinate and downregulate Na(v)1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na(v)1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na(v)1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na(v)1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd 2؉ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd 2؉ on R43C can be obtained with Cd 2؉ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.The epithelial sodium channel (ENaC) 2 and the acid-sensing ion channels (ASICs) in mammals are members of the recently identified ENaC/degenerin family of voltage-insensitive channels (1). The epithelial sodium channel mediates Na ϩ transport in renal and airway-tight epithelia (2). The ASICs are expressed in the central and peripheral nervous system and are possibly involved in nociception, learning, or mechanosensation (3).ENaC and ASICs are likely formed by four homologous subunits. The membrane topology of the channel subunits predicts an amino and a carboxyl terminus facing the inside of the cell, the presence of two transmembrane segments, and a large extracellular loop (4 -7). The structural basis of ENaC/ASIC function remains poorly understood. Amino acid residues in the extracellular loop of ENaC preceding the second transmembrane domain (TM2) bind the pore blocker amiloride (8, 9). Based on previous studies on the interaction between permeant Na ϩ ions and pore blockers of different sizes, it has been proposed that the external pore vestibule of ENaC, where amiloride binds, narrows down to the selectivity filter allowing a Na ϩ or Li ϩ ion to selectively permeate the channel (10). This model is supported by the identification of residues at the external end of ENaC TM2 near the amiloride binding site, which are important for maintaining its high selectivity for Na ϩ (11-15). Beyond the external selectivity filter in TM2, the channel structures lining the ion permeation pathway that are accessible from the cytosol have not yet been identified.In this study, we have used the homomultimeric ASIC1a as a model to investigate the structure of the internal ASIC/ENaC pore. Recently, we have observed that multiple cysteine residues in the amino terminus of ENaC subunits are at least, in part, responsible for the sensitivity of ENaCs to inhibition by intracellular sulfhydryl-modifying agents (16). This suggests that the amino terminus participates in ENaC gating and/or constitutes the internal pore of the channel. In the p...