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.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Renin-aldosterone response, urinary Na/K ratio and growth in pseudohypoaldosteronism patients with mutations in epithelial sodium channel (ENaC) subunit genes

Multi-system pseudohypoaldosteronism (PHA) is a rare syndrome of aldosterone unresponsiveness characterized by symptoms of severe salt-losing caused by mutations in one of the genes that encode alpha, beta or gamma subunit of epithelial sodium channels (ENaC). We examined long-term changes in the renin-aldosterone response in patients with different mutations. Four PHA patients were followed-up for 7-22 years. Patient A with a heterozygous Gly327Cys missense mutation in alphaENaC is a mild case and patients B, C and D are severe cases. Two additional patients with renal PHA served as controls. In patient A, serum aldosterone and plasma renin activity (PRA) decreased with age, PRA reaching near normal values at age 11. In contrast, patients B-D showed a positive correlation between age and aldosterone (r>0.86 for all). In patient B with Arg508 stop mutation, aldosterone reached 166nmol/L at age 19 (>300-fold higher than normal). Urinary Na/K ratios normalized gradually with age in all patients. Growth curves of the patients were reflective of the severity of PHA and compliance with salt therapy. Functional expression studies in oocytes showed that ENaC with alphaGly327Cys mutation, as observed in patient A, showed nearly 40% activity of the wild type ENaC. In contrast, stop mutation as in patient B reduces ENaC activity to less than 5% of the normal. Our results demonstrate distinct genotype-phenotype relationships in multi-system PHA patients. The degree of ENaC function impairment affects differently the renin-aldosterone system and urinary Na/K ratios. The differences observed are age-dependent and PHA form specific.     

Activation of Na+/H+ Exchanger NHE3 by Angiotensin II Is Mediated by Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3 (IRBIT) and Ca2+/Calmodulin-dependent Protein Kinase II

Angiotensin II (ANG II) stimulates renal tubular reabsorption of NaCl by targeting Na⁺/H⁺ exchanger NHE3. We have shown previously that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) plays a critical role in stimulation of NHE3 in response to elevated intracellular Ca²⁺ concentration ([Ca²⁺]_i). In this study, we investigated the role of IRBIT in mediating NHE3 activation by ANG II. IRBIT is abundantly expressed in the proximal tubules where NHE3 is located. ANG II at physiological concentrations stimulates NHE3 transport activity in a model proximal tubule cell line. ANG II-induced activation of NHE3 was abrogated by knockdown of IRBIT, whereas overexpression of IRBIT enhanced the effect of ANG II on NHE3. ANG II transiently increased binding of IRBIT to NHE3 at 5 min but became dissociated by 45 min. In comparison, it took at least 15 min of ANG II treatment for an increase in NHE3 activity and NHE3 surface expression. The stimulation of NHE3 by ANG II was dependent on changes in [Ca²⁺]_i and Ca²⁺/calmodulin-dependent protein kinases II. Inhibition of CaMKII completely blocked the ANG II-induced binding of IRBIT to NHE3 and the increase in NHE3 surface abundance. Several serine residues of IRBIT are thought to be important for IRBIT binding. Mutations of Ser-68, Ser-71, and Ser-74 of IRBIT decreased binding of IRBIT to NHE3 and its effect on NHE3 activity. In conclusion, our current findings demonstrate that IRBIT is critically involved in mediating activation of NHE3 by ANG II via a Ca²⁺/calmodulin-dependent protein kinases II-dependent pathway.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone

Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.     

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.     

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.     

硫酸镍对人红细胞膜Na~+、K~+、Ca~(2+)-ATP酶及乙酰胆碱酯酶活力的影响

实验研究了硫酸镍对人红细胞膜Na+、K+、Ca2+-ATP酶及乙酰胆碱酯酶(AChE)活力的影响,为从分子水平阐明镍毒作用机理及为探索防治措施提供依据。结果表明,硫酸镍对此三种酶均有抑制作用,并呈现较高的量效关系及时效关系。