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• W1992983911 abstract "The degenerin/epithelial Na+ channel (ENaC) superfamily is a group of structurally related ion channels that are involved in diverse biological processes, including responses to mechanical stimuli. In renal cortical collecting ducts, changes in rates of perfusion affect Na+ reabsorption through an amiloride-sensitive pathway, suggesting that ENaC may be a mechanosensitive channel. In this study, we examined whether ENaC expressed in oocytes is regulated by laminar shear stress (LSS). A 1.8-mm (internal diameter) perfusion pipette was placed within 0.5–1.0 mm of the oocyte to provide laminar flow across the oocyte surface. LSS induced a dose-dependent and reversible increase in benzamil-sensitive whole cell Na+ currents in oocytes expressing αβγ ENaC. The half-time for activation by LSS was ∼5 s. Repetitive stimulation by LSS of oocytes expressing ENaC was associated with a reduction in the response to LSS. Oocytes expressing αβS518Kγ, a pore region mutant with a high open probability, were insensitive to LSS. We demonstrated previously that channels with a Cys residue introduced at position αSer-580 had a low open probability, but, following modification by [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), channels exhibited a high open probability. Oocytes expressing αS580Cβγ ENaC respond to LSS similar to wild type; however, covalent modification by MTSET largely eliminated the response to LSS. Our results suggest that shear stress activates ENaC by modifying the gating properties of the channel. The degenerin/epithelial Na+ channel (ENaC) superfamily is a group of structurally related ion channels that are involved in diverse biological processes, including responses to mechanical stimuli. In renal cortical collecting ducts, changes in rates of perfusion affect Na+ reabsorption through an amiloride-sensitive pathway, suggesting that ENaC may be a mechanosensitive channel. In this study, we examined whether ENaC expressed in oocytes is regulated by laminar shear stress (LSS). A 1.8-mm (internal diameter) perfusion pipette was placed within 0.5–1.0 mm of the oocyte to provide laminar flow across the oocyte surface. LSS induced a dose-dependent and reversible increase in benzamil-sensitive whole cell Na+ currents in oocytes expressing αβγ ENaC. The half-time for activation by LSS was ∼5 s. Repetitive stimulation by LSS of oocytes expressing ENaC was associated with a reduction in the response to LSS. Oocytes expressing αβS518Kγ, a pore region mutant with a high open probability, were insensitive to LSS. We demonstrated previously that channels with a Cys residue introduced at position αSer-580 had a low open probability, but, following modification by [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), channels exhibited a high open probability. Oocytes expressing αS580Cβγ ENaC respond to LSS similar to wild type; however, covalent modification by MTSET largely eliminated the response to LSS. Our results suggest that shear stress activates ENaC by modifying the gating properties of the channel. The degenerin (DEG) 1The abbreviations used are: DEGdegenerinENaCepithelial sodium channelLSSlaminar shear stressmENaCmouse ENaCMTSET[2-(trimethylammonium)ethyl]methanethiosulfonate bromideTEVtwo-electrode voltage clamp.1The abbreviations used are: DEGdegenerinENaCepithelial sodium channelLSSlaminar shear stressmENaCmouse ENaCMTSET[2-(trimethylammonium)ethyl]methanethiosulfonate bromideTEVtwo-electrode voltage clamp./epithelial Na+ channel (ENaC) superfamily encodes a group of conserved proteins involved in diverse biological processes, including Na+ homeostasis, salt taste, nociception, touch sensation, and mechanotransduction (1Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (846) Google Scholar, 2Mano I. Driscoll M. BioEssays. 1999; 21: 568-578Crossref PubMed Scopus (135) Google Scholar). DEG/ENaC family members are similar in structure, with intracellular N- and C-termini, two membrane-spanning domains, and a large extracellular region containing cysteine-rich domains. ENaC is formed by three subunits, α, β, and γ, arranged with a subunit stoichiometry of 2α:1β:1γ (3Kosari F. Sheng S. Li J. Mak D.O. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 4Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (368) Google Scholar), although an alternative 3α:3β:3γ stoichiometry has been proposed (5Snyder P.M. Cheng C. Prince L.S. Rogers J.C. Welsh M.J. J. Biol. Chem. 1998; 273: 681-684Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar).Genetic and molecular analyses of Caenorhabditis elegans with a touch-insensitive phenotype led to the identification of several genes whose products were postulated to be part of a touch-transducing complex. Dominant mutations in MEC-4, MEC-10, UNC-8, UNC-105, and DEG-1 result in the deaths of specific mechanosensory neurons where these proteins are expressed, which is thought to result from an increase in cation channel activity (2Mano I. Driscoll M. BioEssays. 1999; 21: 568-578Crossref PubMed Scopus (135) Google Scholar, 6Ernstrom G.G. Chalfie M. Annu. Rev. Genet. 2002; 36: 411-453Crossref PubMed Scopus (143) Google Scholar). Mutations within three distinct regions appear to generate hyperactive channels, including a region following the first transmembrane domain, a tract of 22 amino acids within the extracellular loop present only in degenerins that could be part of a gating domain, and a single amino acid substitution in the pre-second membrane-spanning (M2) domain that appears to be within a conserved gating domain (degenerin site) (2Mano I. Driscoll M. BioEssays. 1999; 21: 568-578Crossref PubMed Scopus (135) Google Scholar). Functional channels were observed when gain-of-function mutations in selected members of the putative touch-transducing complex were expressed in Xenopus oocytes or human embryonic kidney 293 cells, although a functional response to a mechanical stimulus has not been demonstrated (7Garcia-Anoveros J. Garcia J.A. Liu J.D. Corey D.P. Neuron. 1998; 20: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).Cells are potentially exposed to variety of mechanical stimuli, including indentations, high frequency vibrations, osmotic pressure gradients, hydrodynamic pressure, and fluid shear stress. In the specific case of ion channels, modifications in channel kinetics could result from changes in membrane tension, thickness, local curvature, and extension or by direct tugging on the protein by cytoskeletal or extracellular tethers (8Hamill O.P. Martinac B. Physiol. Rev. 2001; 81: 685-740Crossref PubMed Scopus (913) Google Scholar). Based on its structure homology with other members of the DEG/ENaC family that are presumed to be activated by mechanical stimuli (as postulated for degenerins in C. elegans), it was suggested that ENaC could be a mechanosensitive channel (9Awayda M.S. Ismailov I.I. Berdiev B.K. Benos D.J. Am. J. Physiol. 1995; 268: C1450-C1459Crossref PubMed Google Scholar, 10Ismailov I.I. Berdiev B.K. Shlyonsky V.G. Benos D.J. Biophys. J. 1997; 72: 1182-1192Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Various studies have examined this hypothesis with conflicting results. Awayda et al. (9Awayda M.S. Ismailov I.I. Berdiev B.K. Benos D.J. Am. J. Physiol. 1995; 268: C1450-C1459Crossref PubMed Google Scholar) showed that in vitro translated bovine α-ENaC, reconstituted in lipid bilayers, is activated by membrane distension produced by a hydrostatic pressure gradient. Later studies showed that rat αβγ ENaC is mechanosensitive when it is reconstituted in lipid bilayers (10Ismailov I.I. Berdiev B.K. Shlyonsky V.G. Benos D.J. Biophys. J. 1997; 72: 1182-1192Abstract Full Text PDF PubMed Scopus (48) Google Scholar); however conflicting results were reported when oocytes expressing rat ENaC were subjected to an osmotic stimulus (11Awayda M.S. Subramanyam M. J. Gen. Physiol. 1998; 112: 97-111Crossref PubMed Scopus (71) Google Scholar, 12Ji H.L. Fuller C.M. Benos D.J. Am. J. Physiol. 1998; 275: C1182-C1190Crossref PubMed Google Scholar). When channels were examined in principal cells using a cell attached patch configuration, application of negative pressure led to an increase in single channel activity in >30% of patches (13Palmer L.G. Frindt G. J. Gen. Physiol. 1996; 107: 35-45Crossref PubMed Scopus (100) Google Scholar). A recent study suggested that a lack of reproducibility of stretch-mediated ENaC activation may reflect stretch-induced ATP release from cells that inhibits ENaC by activating purinergic receptors, thus masking stretch-induced ENaC activation (14Ma H.P. Li L. Zhou Z.H. Eaton D.C. Warnock D.G. Am. J. Physiol. 2002; 282: F501-F505Crossref Scopus (19) Google Scholar).We recently reported that increasing rates of perfusion of isolated rabbit cortical collecting ducts increase the rates of tubular Na+ reabsorption via an amiloride-sensitive pathway, suggesting that ENaC is activated in response to increases in tubular flow rates (15Satlin L.M. Sheng S. Woda C.B. Kleyman T.R. Am. J. Physiol. 2001; 280: F1010-F1018Crossref PubMed Google Scholar). However, this study did not address the question of whether mechanical forces, such as shear stress, directly modulate gating of the Na+ channel. In this report, we used a submerged jet to expose oocytes expressing ENaC to laminar shear stress (LSS). We observed that wild type ENaC activity increased in response to LSS, whereas mutant or chemically modified Na+ channels that exhibit a high intrinsic open probability did not respond to laminar flow. These data suggest that mechanical forces directly modulate ENaC gating.MATERIALS AND METHODSDNA Constructs—Point mutations and truncations of mouse ENaC (mENaC) subunits were generated by site-directed mutagenesis with the sequential polymerase chain reaction method using Pfu DNA polymerase (Stratagene, La Jolla, CA) (16Sheng S. Li J. McNulty K.A. Avery D. Kleyman T.R. J. Biol. Chem. 2000; 275: 8572-8581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The PCR-amplified fragments containing the desired mutations were ligated into wild type mENaC cDNA. PCR-amplified fragments were sequenced by automated DNA sequencing at the University of Pittsburgh's sequencing facility to confirm the desired mutation.Oocyte Expression—cRNAs for α, β, and γ mENaC subunits were synthesized with T3 mMessage mMachine™ (Ambion, Austin, TX). Stage V-VI Xenopus laevis oocytes were pretreated with 1.5 mg/ml type IV collagenase and injected with 0.5–2 ng/subunit mENaC cRNAs. Injected oocytes were maintained at 18 °C in modified Barth's saline (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 15 mm HEPES, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, and 0.82 mm MgSO4, pH 7.4) supplemented with 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, and 100 μg/ml gentamicin sulfate.Two-electrode Voltage Clamp (TEV)—Two-electrode voltage clamp was performed at room temperature (20–25 °C) using a GeneClamp 500B amplifier (Axon Instruments, Union City, CA). Data were acquired through Clampex 8.0 using a DigiData 1200 interface and stored on the hard disk of a 233 MHz Pentium II personal computer. Pipettes filled with 3 m KCl had resistances of 0.5–5 megaohms. The extracellular solution (TEV solution) was 110 mm NaCl, 2 mm KCl, 1.54 mm CaCl2, and 10 mm HEPES, pH 7.4. Oocytes were maintained in a recording chamber (20-mm diameter and 6-mm-deep Petri dish) with a constant volume of ∼1.5 ml of TEV solution. The recording chamber was perfused at a rate of 3.5 ml/min. Shear stress was applied by perfusing TEV solution through a vertical pipette (1.8-mm internal diameter) that was placed near the surface of the oocyte at a distance of <1 mm as described previously (17Hoger J.H. Ilyin V.I. Forsyth S. Hoger A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7780-7785Crossref PubMed Scopus (116) Google Scholar). Bath perfusion was either maintained (Fig. 2A) or stopped (Figs. 1, 2B, 3, and 4) during the application of LSS. A response to LSS was observed in oocytes when bath perfusion was either maintained or stopped during the application of LSS (see “Results”).Fig. 1ENaC is activated by LSS.A, current-voltage relationship of oocytes expressing αβγ ENaC prior to fluid jet perfusion to generate LSS (closed circle), 1 min following initiation of fluid jet perfusion (open circle), 1 min following discontinuation of fluid jet perfusion (closed triangle), and following the application of 5 μm benzamil in the bath (open triangle) (n = 15). B, representative tracings of shear stress-induced currents. Voltage steps (500 ms) from –140 to 60 mV in 20-mV increments were performed in oocytes expressing wild type ENaC as indicated. C, relative response to LSS of oocytes expressing wild type ENaC. Benzamil-sensitive currents recorded at –100 mV prior to initiation of LSS (before LSS), during stimulation by LSS (+LSS), and following recuperation (after LSS) were plotted as response relative to the mean of the currents recorded before stimulation (Ibasal) (n = 15, p < 0.05; +LSS versus before and after LSS, Kruskal-Wallis non parametric analysis of variance test). D, upper trace, whole cell current recorded from a non-injected oocyte clamped at a holding potential of –90 mV and subjected to stimulation by LSS as indicated by the horizontal bars (+LSS). The tracing is representative of four experiments. Lower trace, current recorded from an oocyte expressing wild type ENaC clamped at a holding potential of –60 mV. The gray bar indicates the addition of benzamil (5 μm) to the bath solution to block currents mediated by ENaC. The black bar indicates the beginning and duration of the vertical jet perfusion. Benzamil (5 μm) was also included in the jet perfusion pipette. Representative of seven experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Constitutively active channels do not respond to LSS.A, representative tracings of experiments performed with oocytes expressing αS580Cβγ. Currents were recorded under control conditions (–LSS) and following 1 min of stimulation by LSS (+LSS). Oocytes were subsequently perfused in the bath with TEV solution containing MTSET (1 mm) for 4 min. The thiol-reactive reagent was washed out by bath perfusion with TEV solution for 8 min. Currents were again recorded before and following 1 min of fluid jet perfusion. The benzamil-insensitive component of the whole cell current was then determined by bath perfusion with TEV solution containing benzamil (5 μm). B, relative responses to LSS of oocytes expressing αS580Cβγ and the degenerin mutant (αβS518Kγ). Currents recorded after 1 min of stimulation by LSS relative to currents recorded before stimulation are shown (I+LSS/I–LSS). Experiments using oocytes expressing αS580Cβγ were performed as described above. The relative response of oocytes expressing αS580Cβγ was not significantly different from that observed with oocytes expressing wild type ENaC (n = 7, p = not significant). However, following covalent modification by MTEST, the relative response to LSS was practically abolished (n = 7, p < 0.05; αS580Cβγ – MTEST (I+LSS/I–LSS) versus αS580Cβγ + MTSET (I+LSS/I-LSS); unpaired t test). In oocytes expressing αβS518Kγ (n = 8), the relative response to LSS was also significantly reduced in comparison with wild type ENaC-expressing oocytes (n = 19, p < 0.01; wild type versus αβS518Kγ, unpaired t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Adaptive response to repetitive mechanical stimulation.A, representative tracing of an experiment performed in an oocyte expressing wild type ENaC and subjected to repetitive stimulation by LSS. The benzamil-insensitive component of whole cell current was subsequently determined by bath perfusion with TEV solution containing benzamil (5 μm). B, same protocol utilized in panel A was performed in an oocyte expressing αR613X, βR564X, and γR583X (αTβTγT) ENaCs. C, relative responses to a single exposure of LSS of oocytes expressing αβγ or αTβTγT. Currents recorded at –100 mV after 1 min of stimulation by LSS relative to currents recorded before stimulation are shown (I+LSS/I–LSS) (n = 10–15, p = not significant). D, relative response of oocytes expressing wild type ENaC (n = 16) or αTβTγT (n = 13) to four repetitive periods of fluid jet perfusion and recuperation. The ratio of the whole cell current measured just prior to and 1 min following the initiation of fluid jet perfusion at a holding potential of –60 mV was determined for each stimulation. Data were normalized to the I+LSS/I–LSS value obtained with the initial fluid jet perfusion. The relative response for oocytes expressing wild type channels (open circles; n = 16) at the second, third, and fourth stimulation were 68 ± 4, 53 ± 5, and 40 ± 5%, respectively. The relative response for oocytes expressing αTβTγT channels (closed squares; n = 13) were 98 ± 7, 82 ± 9, and 80 ± 12%, respectively. Significant differences in the relative response of oocytes expressing wild type ENaC were observed with the second, third, and fourth stimulation, compared with the response of oocytes expressing αTβTγT(p < 0.01; Mann-Whitney test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The approximate average shear stress on the oocyte surface was estimated as the relation of the drag force and the oocyte surface area (Fdrag/surface area of the oocytes) (17Hoger J.H. Ilyin V.I. Forsyth S. Hoger A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7780-7785Crossref PubMed Scopus (116) Google Scholar). The magnitude of the drag force was determined according to Fdrag = 0.5 × ρAω2Cd (where ρ = water density, A = cross-sectional area of the sphere, ω = average free stream velocity, and Cd = drag coefficient (∼1 for spheres with a Reynolds number within the range of ∼3 to ∼25)). The Reynolds number was determined as previously described (17Hoger J.H. Ilyin V.I. Forsyth S. Hoger A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7780-7785Crossref PubMed Scopus (116) Google Scholar). To determine a dose-response relationship, vertical pipette perfusion rates were varied between 0.5 and 3.7 ml/min, corresponding to a shear stress of between 0.013 and 0.753 dynes/cm2. For most studies, the vertical pipette perfusion rate was 1.6 ml/min, corresponding to 0.137 dynes/cm2 of shear stress.Whole cell currents were determined using a series of voltage steps (500 ms) from –140 to 60 mV in 20-mV increments in the absence or presence of benzamil (5 μm) in the bath solution. ENaC-mediated whole cell Na+ currents were defined as the benzamil-sensitive component of the current. Benzamil-sensitive currents measured in the absence of LSS were referred to as I–LSS, whereas currents recorded in the presence of LSS were referred to as I+LSS. To determine mean times of activation, oocytes were clamped at a holding potential of –60 mV, and the vertical pipette perfusion was controlled through the computer using a protocol generated in pClamp 8.0.Data and Statistical Analyses—Data were expressed as the mean ± S.E. (n), unless otherwise indicated, where n equals the number of independent experiments analyzed. Experiments were repeated with a minimum of two batches of oocytes obtained from different frogs. Electrophysiological data were analyzed with Clampfit 8.1 (Axon Instruments, Union City, CA), SigmaPlot 8.02 (SPSS Inc., Chicago, IL), and GraphPad 3.02 (GraphPad Software, San Diego, CA). Statistical comparisons were performed using GraphPad Instant (GraphPad Software, San Diego, CA). Mean time of activation was determined by fitting experimental data with an exponential function y = y0 + a × e–bx, where y0 is the plateau, a is the difference between the signal at time zero and the plateau, and b is the time constant. The mean time of activation was calculated as 0.69302/time constant. The dose-response curve was fit with the following equation y = 1/(1 + 10logEC50–X)), where logEC50 is the logarithm of the dose (shear stress) that elicited 50% of the maximal response.RESULTSLaminar Flow Increases Benzamil-sensitive Na+Currents in Oocytes Expressing αβγ mENaC—We reported previously that the rates of Na+ reabsorption in renal cortical collecting tubules perfused in vitro increase in response to increases in the rates of tubular perfusion (15Satlin L.M. Sheng S. Woda C.B. Kleyman T.R. Am. J. Physiol. 2001; 280: F1010-F1018Crossref PubMed Google Scholar). We also observed increases in whole cell Na+ currents measured in oocytes expressing αβγ mENaC that were centered in a Petri dish in response to a change in the rate of bath perfusion from 0 to 5–6 ml/min, although the increases in currents were highly variable and, on occasion, no increase was observed. To explore whether Na+ channels are activated in response to increases in LSS, we employed a perfusion system similar to the fluid jet used to stimulate sensory hair cells (19Holt J.R. Corey D.P. Eatock R.A. J. Neurosci. 1997; 17: 8739-8748Crossref PubMed Google Scholar). Oocytes expressing αβγ were positioned in the chamber and exposed to laminar fluid flow from a vertical pipette, as described previously by Hoger et al. (17Hoger J.H. Ilyin V.I. Forsyth S. Hoger A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7780-7785Crossref PubMed Scopus (116) Google Scholar). Whole cell Na+ currents were determined before changing from perfusion of the Petri dish to perfusion via a vertical pipette that was placed in close proximity to the oocyte surface. ENaC-mediated Na+ currents, defined as currents sensitive to 5 μm benzamil, were increased significantly following the switch to fluid jet perfusion (Fig. 1, A–C; I/Ibasal = 1.53 ± 0.19, n = 15, p < 0.05; +LSS versus before and after LSS). Channel activation was reversible, as currents fell to baseline levels when perfusion was changed from the vertical pipette back to the bath perfusion (Fig. 1, A–C). Increases in current induced by LSS were not associated with changes in reversal potential, suggesting that LSS did not alter the chemical driving force for Na+ transport (Fig. 1A). Increases in whole cell currents were not observed in non-injected oocytes in response to switching to fluid jet perfusion (Fig. 1D, upper trace). The inclusion of benzamil in the bath solution and vertical pipette abolished the response to LSS in ENaC-expressing oocytes (Fig. 1D, lower trace). Increases in whole cell Na+ currents were also observed if bath perfusion was maintained following initiation of fluid jet perfusion (I+LSS/I–LSS = 1.28 ± 0.08, n = 33). We observed variability in the LSS response (I+LSS/I–LSS) between different batches of oocytes. For that reason, statistical comparisons were made with experiments performed with oocytes from the same batch.A software-controlled perfusion system was utilized to initiate fluid jet perfusion in the setting of constant bath perfusion in order to characterize the kinetics of channel activation. To define the time course of activation by shear stress, whole cell currents were monitored at a clamp potential of –60 mV. Currents were measured with constant bath perfusion for 20 s prior to initiation of fluid jet perfusion. Oocytes expressing αβγ mENaC responded to shear stress with an increase in whole cell currents (Fig. 2A). The time required to achieve half-maximal activation (t½) was obtained from fitting experimental data with a single exponential function. The t½ for activation in response to LSS was 5.3 ± 0.3 s (n = 8). Following channel activation, currents were fairly stable while LSS was maintained for period of 60 s. We did not observe adaptive behavior while LSS was maintained, as was described previously for other mechanosensitive channels (20Hamill O.P. McBride Jr., D.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7462-7466Crossref PubMed Scopus (135) Google Scholar). In some experiments, we observed current rundown that was present before and during stimulation by LSS. Oocytes expressing wild type ENaC were subjected to different amounts of shear stress to determine whether channels responded to increases in shear stress in a dose-dependent manner. Increasing rates of fluid jet perfusion (0 ml/min to 3.7 ml/min, corresponding to a LSS of between ∼0 and 0.733 dynes/cm2) were associated with increases in whole cell Na+ currents in a dose-dependent manner (Fig. 2B).LSS Regulates ENaC by Altering Gating Properties of the Channel—Mechanical stimuli may affect the trafficking of membrane proteins as well as the gating properties of ion channels (8Hamill O.P. Martinac B. Physiol. Rev. 2001; 81: 685-740Crossref PubMed Scopus (913) Google Scholar). To determine whether increases on whole cell Na+ currents in response to LSS occurred in association with increases in channel open probability, we expressed ENaCs with mutations within a gating domain localized to a region preceding the second membrane-spanning (pre-M2) domain of the channel that were previously shown to either increase or decrease channel open probability. αS580Cβγ channels have a low open probability. However, following covalent modification by the sulfhydryl reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate bromide (MTSET), the open probability of this mutant approached a value of 1 (21Sheng S. Li J. McNulty K.A. Kieber-Emmons T. Kleyman T.R. J. Biol. Chem. 2001; 276: 1326-1334Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Oocytes expressing αS580Cβγ were subjected to stimulation by LSS. Increases in whole cell currents in response to shear stress (I+LSS/I–LSS = 1.44 ± 0.12, n = 7 were similar in magnitude to that observed in oocytes expressing wild type ENaC (I+LSS/ I–LSS = 1.46 ± 0.08, n = 19, p = not significant). Following treatment of oocytes expressing αS580Cβγ channels with MTSET (1 mm) to increase channel open probability, the modified channels were insensitive to the shear stress stimulus (Fig. 3, A and B, I+LSS/I–LSS = 1.04 ± 0.08, n = 7, p < 0.05; –MTSET versus +MTSET). A loss of response to shear stress was also observed in oocytes expressing αS580Cβγ that were treated with MTSET (1 mm) prior to their initial exposure to shear stress (I+LSS/I–LSS = 1.01 ± 0.04, n = 5). Previous studies identified a key site (degenerin site) in the pre-M2 domains of ENaC subunits, degenerins, and acid-sensing ion channels where the introduction of bulky residues or the introduction of a Cys residue and subsequent modification by MTS reagents results in channel activation (2Mano I. Driscoll M. BioEssays. 1999; 21: 568-578Crossref PubMed Scopus (135) Google Scholar, 22Snyder P.M. Bucher D.B. Olson D.R. J. Gen. Physiol. 2000; 116: 781-790Crossref PubMed Scopus (77) Google Scholar, 23Adams C.M. Snyder P.M. Price M.P. Welsh M.J. J. Biol. Chem. 1998; 273: 30204-30207Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). ENaCs with a Lys residue substitution at the degenerin site in the β-subunit (corresponding to mouse βS518K) were reported to have a high open probability (22Snyder P.M. Bucher D.B. Olson D.R. J. Gen. Physiol. 2000; 116: 781-790Crossref PubMed Scopus (77) Google Scholar). αβS518Kγ channels were insensitive to LSS (Fig. 3B; I+LSS/I–LSS = 1.07 ± 0.06, n = 8). These data demonstrate that gain-of-function mutations or covalent modifications within a region that dramatically affect channel gating alters the channel's response to mechanical stimulation.To corroborate the proposition that increases in whole cell Na+ currents in response to LSS reflected increases in channel open probability rather than the insertion of channels from an intracellular pool into the plasma membrane, we expressed αS583Cβγ ENaC in oocytes. Covalent modification of this mutant by MTSET (1 mm) reduced whole cell benzamil-sensitive Na+ currents by >90% (I–MTSET = –5.13 ± 0.60 μA and I+MTSET = –0.36 ± 0.09 μA, n = 6). If ENaC stimulation by LSS is due to insertion of channels from an intracellular pool, the increase in benzamil-sensitive Na+ currents in response to LSS in oocytes expressing this mutant before and following covalent modification by MTEST should be similar in magnitude. Oocytes expressing αS583Cβγ responded to LSS with an increase in benzamil-sensitive currents of 0.44 ± 0.04 μA (n = 6). In contrast, the absolute magnitude of the current response to LSS in oocytes modified by MTSET (1 mm) was dramatically reduced to 0.07 ± 0.10 μA(n = 6, p < 0.005) (see Table I). These data suggest that activation of ENaC by LSS is not due to the insertion of channels from an intracellular pool.Table ICovalent modification of α S583Cβ γ abrogated the response to laminar shear stress-LSS IbenzaWhole cell benzamil-sensitive Na+ current.+LSS IbenzaWhole cell benzamil-sensitive Na+ current.ΔIbenzaWhole cell benzamil-sensitive Na+ current.μAμAμA-MTSET (n = 6)bp < 0.005; Mann-Whitney test.-5.13 ± 0.60-5.58 ± 0.61-0.45 ± 0.04+MTSET (n = 6)bp < 0.005; Mann-Whitney test.-0.36 ± 0.09-0.42 ± 0.09-0.06 ± 0.10a Whole cell benzamil-sensitive Na+ current.b p < 0.005; Mann-Whitney test. Open table in a new tab Reduction in the Response to LSS following Repetitive Stimulation—We examined whether ENaCs would respond to repetitive stimulation by LSS. Wild type channels responded to repetit" @default.
• W1992983911 created "2016-06-24" @default.
• W1992983911 creator A5036344246 @default.
• W1992983911 creator A5053328495 @default.
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• W1992983911 date "2004-02-01" @default.
• W1992983911 modified "2023-10-11" @default.
• W1992983911 title "Epithelial Na+ Channels Are Activated by Laminar Shear Stress" @default.
• W1992983911 cites W1538660798 @default.
• W1992983911 cites W1665534701 @default.
• W1992983911 cites W1814044618 @default.
• W1992983911 cites W1820962178 @default.
• W1992983911 cites W1887182106 @default.
• W1992983911 cites W1975465296 @default.
• W1992983911 cites W1992847420 @default.
• W1992983911 cites W1995665478 @default.
• W1992983911 cites W2000998004 @default.
• W1992983911 cites W2020149988 @default.
• W1992983911 cites W2040616883 @default.
• W1992983911 cites W2043191266 @default.
• W1992983911 cites W2044415802 @default.
• W1992983911 cites W2053136734 @default.
• W1992983911 cites W2059727905 @default.
• W1992983911 cites W2061499365 @default.
• W1992983911 cites W2071007358 @default.
• W1992983911 cites W2071843318 @default.
• W1992983911 cites W2074459538 @default.
• W1992983911 cites W2084598544 @default.
• W1992983911 cites W2085966270 @default.
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