FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068761963> ?p ?o ?g. }

• W2068761963 endingPage "9749" @default.
• W2068761963 startingPage "9743" @default.
• W2068761963 abstract "Epithelial Na+ channels (ENaC) participate in the regulation of extracellular fluid volume homeostasis and blood pressure. Channel activity is regulated by both extracellular and intracellular Na+. The down-regulation of ENaC activity by external Na+ is referred to as Na+ self-inhibition. We investigated the structural determinants of Na+ self-inhibition by expressing wild-type or mutant ENaCs in Xenopus oocytes and analyzing changes in whole-cell Na+ currents following a rapid increase of bath Na+ concentration. Our results indicated that wild-type mouse αβγENaC has intrinsic Na+ self-inhibition similar to that reported for human, rat, and Xenopus ENaCs. Mutations at His239 (γH239R, γH239D, and γH239C) in the extracellular loop of the γENaC subunit prevented Na+ self-inhibition whereas mutations of the corresponding His282 in αENaC (αH282D, αH282R, αH282W, and αH282C) significantly enhanced Na+ self-inhibition. These results suggest that these two histidine residues within the extracellular loops are crucial structural determinants for Na+ self-inhibition. Epithelial Na+ channels (ENaC) participate in the regulation of extracellular fluid volume homeostasis and blood pressure. Channel activity is regulated by both extracellular and intracellular Na+. The down-regulation of ENaC activity by external Na+ is referred to as Na+ self-inhibition. We investigated the structural determinants of Na+ self-inhibition by expressing wild-type or mutant ENaCs in Xenopus oocytes and analyzing changes in whole-cell Na+ currents following a rapid increase of bath Na+ concentration. Our results indicated that wild-type mouse αβγENaC has intrinsic Na+ self-inhibition similar to that reported for human, rat, and Xenopus ENaCs. Mutations at His239 (γH239R, γH239D, and γH239C) in the extracellular loop of the γENaC subunit prevented Na+ self-inhibition whereas mutations of the corresponding His282 in αENaC (αH282D, αH282R, αH282W, and αH282C) significantly enhanced Na+ self-inhibition. These results suggest that these two histidine residues within the extracellular loops are crucial structural determinants for Na+ self-inhibition. Epithelial Na+ channels (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; ECL, extracellular loop; Po, open probability; Ki, inhibitory constant; WT, wild type.1The abbreviations used are: ENaC, epithelial Na+ channel; ECL, extracellular loop; Po, open probability; Ki, inhibitory constant; WT, wild type. mediate Na+ transport across apical membranes of high resistance epithelial cells in the kidney, colon, and lung. The regulation of Na+ transport in collecting ducts has an important role in extracellular fluid volume homeostasis and in the control of blood pressure (1Rossier B.C. Pradervand S. Schild L. Hummler E. Annu. Rev. Physiol. 2002; 64: 877-897Crossref PubMed Scopus (316) Google Scholar). Alterations in the functional expression of this channel have been observed in association with several disorders, including inherited forms of hypertension (Liddle syndrome), volume depletion associated with hyperkalemia (pseudohypoaldosteronism type 1), and cystic fibrosis (2Snyder P.M. Endocr. Rev. 2002; 23: 258-275Crossref PubMed Scopus (188) Google Scholar, 3Hummler E. Curr. Hypertens. Rep. 2003; 5: 11-18Crossref PubMed Scopus (46) Google Scholar). Three subunits (α, β, and γ ENaC) have been cloned from different species and are members of the ENaC/degenerin gene superfamily (4Benos D.J. Stanton B.A. J. Physiol. 1999; 520: 631-644Crossref PubMed Scopus (153) Google Scholar). All three subunits participate in formation of the channel pore and share a similar membrane topology with two transmembrane domains (M1 and M2) that are connected by a large extracellular loop (ECL), and cytoplasmic N and C termini (5Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (846) Google Scholar).ENaC activity is regulated by a variety of extracellular and intracellular factors including hormones, proteases, other proteins, as well as monovalent and divalent cations (6Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1033) Google Scholar, 7Schafer J.A. Am. J. Physiol. Renal Physiol. 2002; 283: F221-235Crossref PubMed Scopus (80) Google Scholar). Na+ exerts two distinct types of inhibitory effects on ENaC activity: self-inhibition and feedback inhibition. The inhibitory effects of Na+ are distinguished by their sites of action, modes of action, and time course. Na+ self-inhibition was originally observed in studies of native epithelial tissues in the setting of a rapid increase in extracellular Na+ concentration (6Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1033) Google Scholar, 8Fuchs W. Larsen E.H. Lindemann B. J. Physiol. 1977; 267: 137-166Crossref PubMed Scopus (234) Google Scholar, 9Li J.H. Lindemann B. J. Membr. Biol. 1983; 75: 179-192Crossref PubMed Scopus (42) Google Scholar, 10Turnheim K. Physiol. Rev. 1991; 71: 429-445Crossref PubMed Scopus (70) Google Scholar). It is not dependent on Na+ influx, nor on intracellular Na+ concentration. On the other hand, increases in intracellular Na+ concentration result in feedback inhibition of ENaC (11Komwatana P. Dinudom A. Young J.A. Cook D.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8107-8111Crossref PubMed Scopus (83) Google Scholar, 12Komwatana P. Dinudom A. Young J.A. Cook D.I. J. Membr. Biol. 1998; 162: 225-232Crossref PubMed Scopus (34) Google Scholar, 13Kellenberger S. Gautschi I. Rossier B.C. Schild L. J. Clin. Investig. 1998; 101: 2741-2750Crossref PubMed Scopus (147) Google Scholar, 14Abriel H. Horisberger J.D. J. Physiol. 1999; 516: 31-43Crossref PubMed Scopus (66) Google Scholar). The latter is likely due to a reduction of channel density at the plasma membrane (2Snyder P.M. Endocr. Rev. 2002; 23: 258-275Crossref PubMed Scopus (188) Google Scholar). The inhibition of ENaC by extracellular or intracellular Na+ occurs on different time scales, and may have an important role in limiting increases in intracellular Na+ concentration and cell volume when cells are exposed to physiological conditions that would otherwise dramatically increase rates of cellular Na+ influx via ENaC (10Turnheim K. Physiol. Rev. 1991; 71: 429-445Crossref PubMed Scopus (70) Google Scholar). Both Na+ self-inhibition and feedback inhibition contribute to the complex regulatory network that tightly controls ENaC activity.ENaCs cloned from human, rat, or Xenopus and expressed in Xenopus oocytes exhibit Na+ self-inhibition (15Chraibi A. Horisberger J.D. J. Gen. Physiol. 2002; 120: 133-145Crossref PubMed Scopus (117) Google Scholar). We previously reported that mouse ENaC (mENaC) is blocked by external Ni2+, and identified key extracellular His residues (αHis282 and γHis239) within conserved regions of the α- and γ-subunits that were required for channel block by Ni2+ (16Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). As both Ni2+ and Na+ are extracellular inhibitors of ENaC, we speculated that extracellular regions that participate in Ni2+ block may also have a role in Na+ self-inhibition.We have examined whether specific residues within ENaC ECLs have a role in Na+ self-inhibition by comparing the inhibitory responses to external Na+ of wild-type and mutant mouse αβγENaCs in the oocyte expression system. Wild-type αβγ mENaC displayed typical Na+ self-inhibition. In contrast, we observed that Na+ self-inhibition was dramatically altered in channels containing mutations at either of two His residues within the extracellular loops of α and γ ENaC that dramatically decreased Ni2+ block. Channels with mutations of γHis239 showed no Na+ self-inhibition, whereas channels with a mutation of the corresponding His residue in the α-subunit (αHis282) showed enhanced Na+ self-inhibition.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis—All ENaC clones used in this study were mouse ENaC subunits whose cDNAs were inserted into pBluescript SK-vector (Stratagene, La Jolla, CA) (17Ahn Y.J. Brooker D.R. Kosari F. Harte B.J. Li J. Mackler S.A. Kleyman T.R. Am. J. Physiol. 1999; 277: F121-129Crossref PubMed Google Scholar). Point mutations were generated previously by a PCR-based method (16Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).ENaC Expression and Two Electrode Voltage Clamp—ENaC expression in Xenopus oocytes and two electrode voltage clamp were performed as previously reported (16Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Defoliculated oocytes were injected with 1–4 ng of cRNA for each mENaC subunit (per oocyte) and incubated at 18 °C in modified Barth’s saline (MBS, 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 15mm HEPES, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, 100 μg/ml gentamycin sulfate, pH 7.4). All experiments were performed 20–50 h following cRNA injections at room temperature (20–24 °C). Oocytes were placed in an oocyte recording chamber from Warner Instruments (Hamden, CT) and perfused with constant flow rate of 12–14 ml/min.Procedures for Observing Na+ Self-inhibition—To examine Na+ self-inhibition, a low Na+ bath solution (NaCl-1; containing 1 mm NaCl, 109 mm NMDG, 2 mm KCl, 2 mm CaCl2, 10 mm HEPES, pH 7.4) was replaced rapidly by a high Na+ bath solution (NaCl-110; containing 110 mm NaCl, 2 mm KCl, 2 mm CaCl2, 10 mm HEPES, pH 7.4) while the oocytes were continuously clamped to –60 mV (or –100 mV in some experiments). Bath solution exchange was performed with a 6-channel teflon valve perfusion system from Warner Instruments. At the end of the experiment, 10 μm amiloride was added to the bath to determine the amiloride-insensitive component of the whole cell current. Currents remaining in the presence of 10 μm amiloride were generally less than 200 nA. Results from oocytes that showed unusually large amiloride-insensitive currents (>5% of total currents) were discarded to minimize current contamination from endogenous channels and membrane leak. To ensure consistency, each batch of oocytes injected with wild-type or mutant ENaC cRNAs were examined in an alternating manner.The first 40 s of current decay was fitted with an exponential equation by Clampfit 9.0 (Axon Instruments Inc.). The peak current (Ipeak) was the measured maximal inward current immediately after bath solution exchange from low Na+ to high Na+ concentration. The steady state current (Iss) represented the measured current at 40-s post-Ipeak. The current ratio of Iss/Ipeak was calculated from amiloride-sensitive Iss and Ipeak that were obtained by subtracting amiloride-insensitive currents from Iss and Ipeak.To estimate the Michaelis constants (Km) for Na+ concentration-current relationship, both Ipeak and Iss were measured in the same cell after the bath Na+ concentration was raised from 1 to 3, 10, 30, 60, and 110 mm and were normalized to their maximal values. The relative Ipeak and Iss were plotted against the Na+ concentrations. Km and Vmax (maximal relative current) were obtained by best fitting of the current-concentration data according to Equation 1 with least square non-linear curve fitting using Origin Pro 7.0 (OriginLab Corporation, Northampton, MA). I=Vmax·C/(C+Km)(Eq. 1) In the above equation, I is the relative Ipeak or Iss and the C refers to the Na+ concentration used to initiate self-inhibition.We estimated the apparent inhibitory constant (Ki) of Na+ self-inhibition from the results of the above experiments. The ratios of Iss and Ipeak representing the amplitudes of self-inhibition at the different Na+ concentrations were plotted against the external Na+ concentrations. A Ki value was estimated from a best fitting of the data with Hill (Equation 2). ISS/Ipeak=Kin/(Cn+Kin)(Eq. 2) In Equation 2, C is the Na+ concentration and the n is the Hill coefficient.Statistical Analysis—Data are presented as mean ± S.E. Significance comparisons between groups were performed with unpaired Student’s t tests. A p value of less than 0.05 was considered statistically different.RESULTSNa+ Self-inhibition of αβγ mENaC—We examined Na+ self-inhibition of αβγ mENaC expressed in Xenopus oocytes by tracking the current decay following a rapid increase in the extracellular Na+ concentration from 1 to 110 mm. The inward current measured at –60 mV quickly reached its maximal level (Ipeak) and then relaxed exponentially to a relatively steady level (Iss) (Fig. 1). Two parameters were used to describe the speed and amplitude of Na+ self-inhibition, the time constant (τ) for the current decline and the ratio of the steady state (Iss) to peak (Ipeak) whole cell amiloride-sensitive Na+ current. Current decay was observed with a time constant of 8.14 ± 0.38 s (n = 20) (Fig. 1). The ratio of steady state current to peak current was 0.66 ± 0.02 (n = 20). The time course and amplitude of the self-inhibition we observed in αβγ mENaC are comparable, though not identical, to those reported for human, rat and Xenopus αβγ ENaCs (15Chraibi A. Horisberger J.D. J. Gen. Physiol. 2002; 120: 133-145Crossref PubMed Scopus (117) Google Scholar, 18Babini E. Geisler H.S. Siba M. Grunder S. J. Biol. Chem. 2003; 278: 28418-28426Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Na+ self-inhibition did not appear to exhibit voltage-dependence, as we observed similar responses at clamping voltages of –40, –60, –80, and –100 mV. 2S. Sheng, J. B. Bruns, and T. R. Kleyman, unpublished data. A similar self-inhibition response was seen in oocytes expressing different levels of currents, varying from –1 to –20 μA at a holding potential of –60 mV. The intracellular Na+ concentration did not significantly affect the speed or amplitude of Na+ self-inhibition, as the responses were similar in Na+-loaded oocytes that were incubated in 88 mm Na+ MBS and in oocytes that were kept in 10 mm Na+ MBS following cRNA injection. It has been reported that Na+ self-inhibition is not dependent on the current level, nor on intracellular Na+ concentration (15Chraibi A. Horisberger J.D. J. Gen. Physiol. 2002; 120: 133-145Crossref PubMed Scopus (117) Google Scholar).Substitutions of γHis239 within the Extracellular Loop Eliminated Na+ Self-inhibition—One of the peculiar features of ENaC architecture is that each subunit possesses a very large extracellular loop composed of about 450 residues. While its role in the regulation of channel activity is still unclear, recent studies suggest that the ECL may be involved in inhibitor binding (amiloride and Ni2+), channel gating, and subunit assembly and processing (19Ismailov II Kieber-Emmons T. Lin C. Berdiev B.K. Shlyonsky V.G. Patton H.K. Fuller C.M. Worrell R. Zuckerman J.B. Sun W. Eaton D.C. Benos D.J. Kleyman T.R. J. Biol. Chem. 1997; 272: 21075-21083Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Kelly O. Lin C. Ramkumar M. Saxena N.C. Kleyman T.R. Eaton D.C. Am. J. Physiol. Renal. Physiol. 2003; 285: F1279-F1290Crossref PubMed Scopus (34) Google Scholar, 21Firsov D. Robert-Nicoud M. Gruender S. Schild L. Rossier B.C. J. Biol. Chem. 1999; 274: 2743-2749Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 23Bruns J.B. Hu B. Ahn Y.J. Sheng S. Hughey R.P. Kleyman T.R. Am. J. Physiol. Renal. Physiol. 2003; 285: F600-F609Crossref PubMed Scopus (22) Google Scholar). A six residue tract (WYRFHY) within the ECL of the α-subunit was identified as a putative amiloride binding site based on its homology with the antigen binding domain of an anti-amiloride antibody (19Ismailov II Kieber-Emmons T. Lin C. Berdiev B.K. Shlyonsky V.G. Patton H.K. Fuller C.M. Worrell R. Zuckerman J.B. Sun W. Eaton D.C. Benos D.J. Kleyman T.R. J. Biol. Chem. 1997; 272: 21075-21083Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 24Kieber-Emmons T. Lin C. Foster M.H. Kleyman T.R. J. Biol. Chem. 1999; 274: 9648-9655Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Specific mutations within this tract altered the sensitivity of α-subunit channels to amiloride, and altered gating characteristics of both α channels as well as αβγ channels (16Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19Ismailov II Kieber-Emmons T. Lin C. Berdiev B.K. Shlyonsky V.G. Patton H.K. Fuller C.M. Worrell R. Zuckerman J.B. Sun W. Eaton D.C. Benos D.J. Kleyman T.R. J. Biol. Chem. 1997; 272: 21075-21083Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Kelly O. Lin C. Ramkumar M. Saxena N.C. Kleyman T.R. Eaton D.C. Am. J. Physiol. Renal. Physiol. 2003; 285: F1279-F1290Crossref PubMed Scopus (34) Google Scholar). We and other investigators (16Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 25Segal A. Cucu D. Van Driessche W. Weber W.M. FEBS Lett. 2002; 515: 177-183Crossref PubMed Scopus (25) Google Scholar) recently reported that Ni2+ is an external inhibitor of ENaC expressed in Xenopus oocytes. The αHis282 within this tract and the corresponding residue in the γ-subunit (γHis239) were identified as putative Ni2+ binding sites (16Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). As both external Ni2+ and high concentrations of extracellular Na+ inhibit ENaC, we hypothesized that they might share a similar mechanism for their inhibitory effects on ENaC. This prompted us to examine the possibility that sites responsible for Ni2+ block may participate in Na+ self-inhibition. We examined Na+ self-inhibition of mutant mENaCs containing point mutations at either αH282 or γH239. To our surprise, Na+ self-inhibition was not observed in oocytes expressing αβγH239R, αβγH239D, or αβγH239C mENaC. As shown in Figs. 1C and 2, the currents reached their maximal level and essentially stayed at the same level, with minimal loss of current over a 1-min period.Fig. 2Mutations at γHis239 eliminated Na+ self-inhibition. Representative recordings from oocytes expressing αβγH239R (A), αβγH239D (B), or αβγH239C (C) mENaCs are shown. Identical responses of the whole cell currents to rapid increases in the external Na+ concentration were observed in at least 10 oocytes for each mutant. Open and gray bars indicate the time when the cells were bathed in 1 and 110 mm Na+ solutions, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutations of αHis282 Enhanced Na+ Self-inhibition—We observed that several αH282 mutants that were examined, including αH282D, αH282R, and αH282C, enhanced Na+ self-inhibition as shown by significantly smaller τ and Iss/Ipeak values than those of wild type (Fig. 3 and Table I). Although the response of αH282Wβγ mENaC was significantly faster than wild type, the Iss/Ipeak was similar to wild type (Fig. 3 and Table I).Fig. 3Mutations at αHis282 increased Na+ affinity for self-inhibition. Na+ self-inhibition was examined in individual oocytes expressing wild-type or mutant channels with extracellular Na+ concentrations of 110, 60, 30, 10, or 3 mm. A, recordings of current changes in response to the alterations of external Na+ concentration in oocytes expressing αβγ, αH282Dβγ, αH282Rβγ, αH282Wβγ, or αH282Cβγ, were representative of at least four experiments for each channel type. The short black lines above each trace indicate the periods of time when the cell was bathed in a 1 mm NaCl solution. The numbers below each current decay are the Na+ concentrations (in mm) used for inducing self-inhibition. B, relationship of the relative Ipeak and external Na+ concentrations. Relative Ipeak represents the individual peak current at a specific Na+ concentration that was normalized to the maximal Ipeak observed in the same cell. Lines are from the best fitting with Equation 1. C, relationship of the relative Iss (normalized to maximal Iss measured in the same cell) and external Na+ concentration. Solid lines are from best fitting with Equation 1 and dashed lines are from the best fitting with Equation 3. D, the relationship of the ratio of Iss and Ipeak and Na+ concentrations. Lines are from the best fitting of the data with the Hill equation. E, the relationship of the time constants of Na+ self-inhibition and Na+ concentrations (30, 60, and 110 mm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ICharacteristics of Na+ self-inhibition in wild-type and mutant mENaCsmENaCOocytesτIpeakIssIss/IpeaksμAμAαβγ208.14 ± 0.38-4.89 ± 0.79-3.23 ± 0.500.66 ± 0.02αH282Dβγ193.32 ± 0.16aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.-4.98 ± 0.51-1.79 ± 0.180.37 ± 0.03aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αH282Rβγ104.34 ± 0.22aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.-4.37 ± 0.83-1.66 ± 0.310.38 ± 0.01aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αH282Wβγ44.30 ± 0.79aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.-2.52 ± 0.55-1.86 ± 0.630.69 ± 0.08αH282Cβγ65.17 ± 0.33aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.-3.18 ± 0.29-1.33 ± 0.130.43 ± 0.04aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αβQ220Hγ68.17 ± 0.57-2.31 ± 0.29-1.48 ± 0.200.64 ± 0.02αβγH239R6> 40-4.75 ± 1.60-4.64 ± 1.560.99 ± 0.01aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αβγH239D5> 40-2.76 ± 1.04-2.61 ± 0.970.96 ± 0.02aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αH282RβγH239R616.87 ± 0.95aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.-1.86 ± 0.35-1.67 ± 0.310.90 ± 0.00aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.,bValue is p < 0.001 from Student’s t test between αH282RβγH239R and αβγH239R.αS580Cβγ105.46 ± 0.19aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.-6.30 ± 0.19-3.70 ± 0.180.58 ± 0.02cValue is p < 0.01 from Student’s t test between WT(αβγ) and mutant channels.αH282R-S580Cβγ62.61 ± 0.10aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.,dValues are p < 0.01 from Student’s t test between αH282R-S580Cβγ and αH282Rβγ.,eValues are p < 0.001 from Student’s t test between αH282R-S580Cβγ and αS580Cβγ.-5.30 ± 0.51-1.66 ± 0.140.32 ± 0.01aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.,dValues are p < 0.01 from Student’s t test between αH282R-S580Cβγ and αH282Rβγ.,eValues are p < 0.001 from Student’s t test between αH282R-S580Cβγ and αS580Cβγ.a Values are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.b Value is p < 0.001 from Student’s t test between αH282RβγH239R and αβγH239R.c Value is p < 0.01 from Student’s t test between WT(αβγ) and mutant channels.d Values are p < 0.01 from Student’s t test between αH282R-S580Cβγ and αH282Rβγ.e Values are p < 0.001 from Student’s t test between αH282R-S580Cβγ and αS580Cβγ. Open table in a new tab To further characterize the enhanced Na+ self-inhibition by these mutations, we performed additional experiments to determine the apparent Na+ affinity of wild-type and mutant channels. We examined self-inhibition responses in a repetitive manner in the same oocyte, as the speed (τ) and extent of inhibition (Iss/Ipeak) remained constant, despite slowly decreasing overall whole cell Na+ currents, as has been reported by others (15Chraibi A. Horisberger J.D. J. Gen. Physiol. 2002; 120: 133-145Crossref PubMed Scopus (117) Google Scholar). The response of individual oocytes to acute increases in the concentration of external Na+ from 1 mm to 110, 60, 30, 10, and 3 mm was measured in order to estimate an apparent Na+ affinity for self-inhibition. As seen in Fig. 3A, the self-inhibition responses in wild-type channels were faster and deeper with 110 mm Na+ than with 60 or 30 mm Na+. When external Na+ was increased from 1 to 3 or 10 mm, no self-inhibition was observed. The estimated Ki for Na+ self-inhibition was 210 ± 19 mm (n = 6, Table II). These results are consistent with the notion that Na+ self-inhibition is a low affinity process (15Chraibi A. Horisberger J.D. J. Gen. Physiol. 2002; 120: 133-145Crossref PubMed Scopus (117) Google Scholar). On the contrary, currents from oocytes expressing the mutant channels containing αH282D, αH282R, αH282W, or αH282C showed more rapid and deeper (except for αH282W) current relaxations than wild type with 110, 60, and 30 mm Na+. In addition, self-inhibition was apparent in oocytes expressing αH282Dβγ and αH282Rβγ when external Na+ was increased from 1 to 10 mm Na+. The estimated Ki values for αH282D, αH282R, and αH282C channels were 62, 76, and 87 mm, respectively, significantly lower than that of wild type.Table IIFitting parameters for the dose responses of Na+ self-inhibitionmENaCOocytesIpeakIssIssIssIss/IpeakKmKmKmKiKimmαβγ636.39 ± 6.8219.76 ± 3.3045.89 ± 7.12142.40 ± 21.83210.71 ± 18.91αH282Dβγ928.37 ± 3.037.55 ± 0.86aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.25.15 ± 3.51bValues are p < 0.05 from Student’s t test between WT(αβγ) and mutant channels.85.66 ± 12.45bValues are p < 0.05 from Student’s t test between WT(αβγ) and mutant channels.61.70 ± 6.21aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αH282Rβγ638.48 ± 4.199.65 ± 1.42bValues are p < 0.05 from Student’s t test between WT(αβγ) and mutant channels.40.53 ± 8.7276.92 ± 14.56bValues are p < 0.05 from Student’s t test between WT(αβγ) and mutant channels.75.50 ± 4.45aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αH282Cβγ547.75 ± 4.4813.23 ± 0.6690.18 ± 10.09cValues are p < 0.01 from Student’s t test between WT(αβγ) and mutant channels.32.96 ± 4.00cValues are p < 0.01 from Student’s t test between WT(αβγ) and mutant channels.87.14 ± 8.41aValues are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.αH282Wβγ445.96 ± 6.2527.04 ± 5.05NDdNot determined.ND259.34 ± 153.51a Values are p < 0.001 from Student’s t test between WT(αβγ) and mutant channels.b Values are p < 0.05 from Student’s t test between WT(αβγ) and mutant channels.c Values are p < 0.01 from Student’s t test between WT(αβγ) and mutant channels.d Not determined. Open table in a new tab The relationships of the Ipeak and Iss versus Na+ concentration (greater than 1 mm) for wild-type channels were fit with the Michaelis-Menten equation (Equation 1), generating an apparent Km for Na+ of 36 ± 7 mm and 20 ± 3 mm for Ipeak and Iss, respectively (Table II and Fig. 3, B and C). Peak current Km values of the αHis282 mutants were similar to wild type. However, αH282D and αH282R significantly reduced the apparent Iss Km. Curve fitting of Ipeak versus Na+ concentration with Equation 1 showed an average correlation coefficient of 0.979; whereas the fitting of Iss versus Na+ concentration was less satisfactory with an average correlation coefficient of 0.935 for wild type, and correlation coefficients of 0.822, 0.837, and 0.878 for mutant channels containing αH282D, αH282R, or αH282C, respectively. This poor fitting reflects reductions in whole cell currents observed with Na+ concentrations greater than 30 mm due to Na+ self-inhibition (Fig. 3C). As the response of these mutant channels to acute increases in external Na+ concentration appeared to be similar to substrate inhibition of an enzyme, we analyzed our data with Equation 3 that describes substrate inhibition (26Schulz A.R. Enzyme Kinetics: From Disease to Multienzyme Systems. Cambridge University Press, Cambridge, UK1994: 38-41Google Scholar), Iss=Vmax·C/(Km+C+C2/Ki)(Eq. 3) where Ki is an inhibitory constant for Na+ that reflects Na+ self-inhibition. Curve fitting of Iss versus Na+ concentration with Equation 3 showed average correlation coefficients of greater than 0.97 for both wild-type and mutant channels. The estimated Ki values for wild-type and mutant channels (H282D, αH282R, and αH282C) with Equation 3 were comparable to those obtained by fitting the Iss/Ipeak data with Hill equation (see Equation 2 and see Table II). The results suggest that substitution of αHis282 with Asp, Arg, or Cys increased apparent affinity of Na+ for self-inhibition.The estimated Km values for channels containing mutations at γHis239 were in the range of 72 to 100 mm, much higher than that of wild-type ENaC. This observation suggested that current saturation of wild-type ENaC (particularly for Iss) at low Na+ concentrations was the result of Na+ self-inhibition.As mutations of αHis282 and γHis239 resulted in opposite effects on Na+ self-inhibition, we examined the self-inhibition response of a channel with mutations at both sites (αH282R-β-γH239R mENaC). Oocytes expressing these channels responded to an increase in the bath Na+ concentration from 1 to 110 mm with a" @default.
• W2068761963 created "2016-06-24" @default.
• W2068761963 creator A5036344246 @default.
• W2068761963 creator A5073513460 @default.
• W2068761963 creator A5088195948 @default.
• W2068761963 date "2004-03-01" @default.
• W2068761963 modified "2023-10-17" @default.
• W2068761963 title "Extracellular Histidine Residues Crucial for Na+ Self-inhibition of Epithelial Na+ Channels" @default.
• W2068761963 cites W1820962178 @default.
• W2068761963 cites W1969651682 @default.
• W2068761963 cites W1971849369 @default.
• W2068761963 cites W1981924141 @default.
• W2068761963 cites W1983524637 @default.
• W2068761963 cites W1992847420 @default.
• W2068761963 cites W1993363645 @default.
• W2068761963 cites W1996447493 @default.
• W2068761963 cites W2012396734 @default.
• W2068761963 cites W2015005230 @default.
• W2068761963 cites W2017334692 @default.
• W2068761963 cites W2018499368 @default.
• W2068761963 cites W2021097521 @default.
• W2068761963 cites W2028495056 @default.
• W2068761963 cites W2031839150 @default.
• W2068761963 cites W2034728504 @default.
• W2068761963 cites W2037737642 @default.
• W2068761963 cites W2040616883 @default.
• W2068761963 cites W2048419229 @default.
• W2068761963 cites W2051712209 @default.
• W2068761963 cites W2058804065 @default.
• W2068761963 cites W2060838963 @default.
• W2068761963 cites W2061930829 @default.
• W2068761963 cites W2064265775 @default.
• W2068761963 cites W2065864299 @default.
• W2068761963 cites W2071007358 @default.
• W2068761963 cites W2072079878 @default.
• W2068761963 cites W2076541507 @default.
• W2068761963 cites W2085966270 @default.
• W2068761963 cites W2096976107 @default.
• W2068761963 cites W2100317999 @default.
• W2068761963 cites W2106392794 @default.
• W2068761963 cites W2108919815 @default.
• W2068761963 cites W2110572874 @default.
• W2068761963 cites W2115009943 @default.
• W2068761963 cites W2120245762 @default.
• W2068761963 cites W2172109574 @default.
• W2068761963 cites W2182013323 @default.
• W2068761963 cites W2184212489 @default.
• W2068761963 cites W2409568467 @default.
• W2068761963 cites W83227328 @default.
• W2068761963 doi "https://doi.org/10.1074/jbc.m311952200" @default.
• W2068761963 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14701851" @default.
• W2068761963 hasPublicationYear "2004" @default.
• W2068761963 type Work @default.
• W2068761963 sameAs 2068761963 @default.
• W2068761963 citedByCount "78" @default.
• W2068761963 countsByYear W20687619632012 @default.
• W2068761963 countsByYear W20687619632013 @default.
• W2068761963 countsByYear W20687619632014 @default.
• W2068761963 countsByYear W20687619632015 @default.
• W2068761963 countsByYear W20687619632016 @default.
• W2068761963 countsByYear W20687619632017 @default.
• W2068761963 countsByYear W20687619632018 @default.
• W2068761963 countsByYear W20687619632019 @default.
• W2068761963 countsByYear W20687619632020 @default.
• W2068761963 countsByYear W20687619632021 @default.
• W2068761963 countsByYear W20687619632022 @default.
• W2068761963 countsByYear W20687619632023 @default.
• W2068761963 crossrefType "journal-article" @default.
• W2068761963 hasAuthorship W2068761963A5036344246 @default.
• W2068761963 hasAuthorship W2068761963A5073513460 @default.
• W2068761963 hasAuthorship W2068761963A5088195948 @default.
• W2068761963 hasBestOaLocation W20687619631 @default.
• W2068761963 hasConcept C12554922 @default.
• W2068761963 hasConcept C185592680 @default.
• W2068761963 hasConcept C2778460671 @default.
• W2068761963 hasConcept C28406088 @default.
• W2068761963 hasConcept C515207424 @default.
• W2068761963 hasConcept C55493867 @default.
• W2068761963 hasConcept C86803240 @default.
• W2068761963 hasConcept C95444343 @default.
• W2068761963 hasConceptScore W2068761963C12554922 @default.
• W2068761963 hasConceptScore W2068761963C185592680 @default.
• W2068761963 hasConceptScore W2068761963C2778460671 @default.
• W2068761963 hasConceptScore W2068761963C28406088 @default.
• W2068761963 hasConceptScore W2068761963C515207424 @default.
• W2068761963 hasConceptScore W2068761963C55493867 @default.
• W2068761963 hasConceptScore W2068761963C86803240 @default.
• W2068761963 hasConceptScore W2068761963C95444343 @default.
• W2068761963 hasIssue "11" @default.
• W2068761963 hasLocation W20687619631 @default.
• W2068761963 hasOpenAccess W2068761963 @default.
• W2068761963 hasPrimaryLocation W20687619631 @default.
• W2068761963 hasRelatedWork W1540690193 @default.
• W2068761963 hasRelatedWork W160183570 @default.
• W2068761963 hasRelatedWork W1748687783 @default.
• W2068761963 hasRelatedWork W1933973922 @default.
• W2068761963 hasRelatedWork W1970082812 @default.
• W2068761963 hasRelatedWork W2051885497 @default.

• next