Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

The voltage-gated K⁺ (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na⁺ and Ca²⁺ currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na⁺ (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.     

Targeted proteomics using immunoblotting technique for studying dysregulation of ion transporters in renal disorders

Renal salt and water transport physiology has benefited tremendously from the rapid advance of proteomics. Proteomics developed as a fast-throughput means of screening for global changes in proteins in a selected tissue, organ or cell type, as a logical offshoot of similar comprehensive, messenger RNA array-type technology. Targeted proteomics utilizes similar techniques but examines a predetermined set of proteins. One approach that has been rigorously employed over the last 10 years to evaluate differences in renal protein abundances due to a treatment or genotype has been parallel semiquantitative immunoblotting using antibody arrays. This approach, and newer ones on the horizon, provide a rapid global overview of regulation of the individual proteins whose integrated action determines overall renal sodium or water reabsorption.     

Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel

Ubiquitination plays a key role in trafficking of the epithelial Na⁺ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na⁺ absorption.     

Enhanced expression of the Epithelial Sodium Channel in neutrophils from hypertensive patients

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules.Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process.     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.     

Lipid modulation of ion channels through specific binding sites

Ion channel conformational changes within the lipid membrane are a key requirement to control ion passage. Thus, it seems reasonable to assume that lipid composition should modulate ion channel function. There is increasing evidence that this implicates not just an indirect consequence of the lipid influence on the physical properties of the membrane, but also specific binding of selected lipids to certain protein domains. The result is that channel function and its consequences on excitability, contractility, intracellular signaling or any other process mediated by such channel proteins, could be subjected to modulation by membrane lipids. From this it follows that development, age, diet or diseases that alter lipid composition should also have an influence on those cellular properties. The wealth of data on the non-annular lipid binding sites in potassium channel from Streptomyces lividans (KcsA) makes this protein a good model to study the modulation of ion channel structure and function by lipids. The fact that this protein is able to assemble into clusters through the same non-annular sites, resulting in large changes in channel activity, makes these sites even more interesting as a potential target to develop lead compounds able to disrupt such interactions and hopefully, to modulate ion channel function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na⁺ absorption. Although colonic Na⁺ absorption is mediated by both epithelial Na⁺ channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na⁺ absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na⁺ absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na⁺ fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO₃⁻-dependent Na⁺ absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption, whereas DSS-induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO₃⁻-dependent) Na⁺ absorption. In in vivo loop studies HCO₃⁻-Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, whereas in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO₃⁻-Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-mediated Na⁺ absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.     

Acetylation Stimulates the Epithelial Sodium Channel by Reducing Its Ubiquitination and Degradation

The epithelial Na⁺ channel (ENaC) functions as a pathway for Na⁺ absorption in the kidney and lung, where it is crucial for Na⁺ homeostasis and blood pressure regulation. ENaC is regulated in part through signaling pathways that control the ubiquitination state of ENaC lysines. A defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. Here we determined that α-, β-, and γENaC are also substrates for lysine acetylation. Trichostatin A (TSA), a histone deacetylase inhibitor, enhanced ENaC acetylation and increased ENaC abundance in the total cell lysate and at the cell surface. Moreover, TSA increased ENaC current in Fischer rat thyroid and kidney collecting duct epithelia. We found that HDAC7 is expressed in the kidney collecting duct, supporting a potential role for this histone deacetylase in ENaC regulation. HDAC7 overexpression reduced ENaC abundance and ENaC current, whereas ENaC abundance and current were increased by silencing of HDAC7. ENaC and HDAC7 form a complex, as detected by coimmunoprecipitation. We observed a reciprocal relationship between acetylation and ubiquitination; TSA reduced ENaC ubiquitination, whereas HDAC7 increased ubiquitination. By reducing ENaC ubiquitination, TSA decreased the rate of ENaC degradation. Thus, acetylation increases epithelial Na⁺ absorption by antagonizing ENaC ubiquitination. This stabilizes ENaC, and hence, increases its abundance at the cell surface.