On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+ Channel Binding Site
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m503500200
ISSN1083-351X
AutoresOssama B. Kashlan, Shaohu Sheng, Thomas R. Kleyman,
Tópico(s)Ion Channels and Receptors
ResumoThe epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are αSer-583, βGly-525, and γGly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the β and γ glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the α subunit resulted in moderate (<5-fold) weakening of amiloride Ki. We investigated the role of αSer-583 in amiloride binding by systematically mutating αSer-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a β-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at α583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some αSer-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that αSer-583 may have a role in ion permeation through ENaC. The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are αSer-583, βGly-525, and γGly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the β and γ glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the α subunit resulted in moderate (<5-fold) weakening of amiloride Ki. We investigated the role of αSer-583 in amiloride binding by systematically mutating αSer-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a β-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at α583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some αSer-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that αSer-583 may have a role in ion permeation through ENaC. The epithelial sodium channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; wt, wild type; I-V, current-voltage. is the primary target of the potassium-sparing diuretic, amiloride. ENaC is expressed in the apical membrane of sodium-absorptive epithelia such as the distal nephron, lung airway and alveoli, and descending colon (1Li X.J. Blackshaw S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1814-1818Crossref PubMed Scopus (56) Google Scholar). As such, ENaC plays a critical role in maintaining Na+ homeostasis, controlling blood pressure and airway fluid volume. ENaC is a member of the ENaC/Degenerin superfamily, all of which conduct Na+, are inhibited by amiloride, and have some structural features in common. They each have two hydrophobic membrane-spanning domains (M1 and M2) with intracellular N and C termini and a large extracellular loop containing two or three cysteine-rich domains (2Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (864) Google Scholar). To date, α, β, γ, and δ ENaC subunits, which share 30–40% sequence identity, have been identified in mammals. ENaC is largely found in the kidney, lung, and colon as comprised of α, β, and γ subunits, although the subunit stoichiometry of functional channels remains controversial. Several groups have proposed a tetrameric α2βγ quaternary structure, although a nonameric α3β3γ3 arrangement has also been proposed (3Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (370) Google Scholar, 4Kosari 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 (202) Google Scholar, 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 (207) Google Scholar, 6Staruschenko A. Medina J.L. Patel P. Shapiro M.S. Booth R.E. Stockand J.D. J. Biol. Chem. 2004; 279: 27729-27734Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The δ subunit may substitute for α in some tissues. Understanding the mode of amiloride binding and the mechanism by which it inhibits ENaC has been the objective of numerous studies (2Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (864) Google Scholar). Apparent amiloride binding affinity was found to be attenuated by mutations at several specific sites. Mutations at αSer-583, βGly-525, and γGly-542 (γGly-537 using rat ENaC numbering), which are located in a region preceding the second membrane-spanning domain, altered amiloride Ki (7Ji H.L. Bishop L.R. Anderson S.J. Fuller C.M. Benos D.J. J. Biol. Chem. 2004; 279: 8428-8440Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 8Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (240) Google Scholar, 9Kellenberger S. Gautschi I. Schild L. Mol. Pharmacol. 2003; 64: 848-856Crossref PubMed Scopus (70) Google Scholar). Other sites were identified that localized to the extracellular loop of the α subunit (10Ismailov I.I. 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, 11Kelly O. Lin C. Ramkumar M. Saxena N.C. Kleyman T.R. Eaton D.C. Am. J. Physiol. 2003; 285: F1279-F1290Crossref PubMed Scopus (34) Google Scholar, 12Li X.J. Xu R.H. Guggino W.B. Snyder S.H. Mol. Pharmacol. 1995; 47: 1133-1140PubMed Google Scholar, 13Kieber-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). Two general mechanisms, ionic block and allostery, have been proposed for the inhibition of ENaC currents by amiloride (14Garty H. Benos D.J. Physiol. Rev. 1988; 68: 309-373Crossref PubMed Scopus (314) Google Scholar). Support of the ionic block model was grounded on the findings that amiloride block was voltage sensitive and competitive with Na+ in certain species and that amiloride may bind to the extracellular mouth of the pore (7Ji H.L. Bishop L.R. Anderson S.J. Fuller C.M. Benos D.J. J. Biol. Chem. 2004; 279: 8428-8440Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 8Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (240) Google Scholar, 9Kellenberger S. Gautschi I. Schild L. Mol. Pharmacol. 2003; 64: 848-856Crossref PubMed Scopus (70) Google Scholar, 15Hamilton K.L. Eaton D.C. Membr. Biochem. 1986; 6: 149-171Crossref PubMed Scopus (53) Google Scholar, 16Palmer L.G. J. Membr. Biol. 1984; 80: 153-165Crossref PubMed Scopus (84) Google Scholar). Support of the allostery model derived from evidence that amiloride may interact with nonpore-associated regions within ENaC, is non-competitive with Na+ in certain species, and some amiloride analogues stimulate Na+ current (10Ismailov I.I. 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, 11Kelly O. Lin C. Ramkumar M. Saxena N.C. Kleyman T.R. Eaton D.C. Am. J. Physiol. 2003; 285: F1279-F1290Crossref PubMed Scopus (34) Google Scholar, 12Li X.J. Xu R.H. Guggino W.B. Snyder S.H. Mol. Pharmacol. 1995; 47: 1133-1140PubMed Google Scholar, 13Kieber-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, 17Benos D.J. Mandel L.J. Balaban R.S. J. Gen. Physiol. 1979; 73: 307-326Crossref PubMed Scopus (70) Google Scholar, 18Benos D.J. Mandel L.J. Simon S.A. J. Membr. Biol. 1980; 56: 149-158Crossref PubMed Scopus (22) Google Scholar, 19Li J.H. Lindemann B. J. Membr. Biol. 1983; 75: 179-192Crossref PubMed Scopus (42) Google Scholar). In this study, we sought to understand the role, if any, of the αSer-583 side chain in binding amiloride. Previously, mutations at αSer-583 have been found to cause only modest changes in apparent amiloride binding affinity, in stark contrast to all mutations made at the analogous positions in the β and γ subunits (βGly-525 and γGly-542) where any mutation of the Gly residue greatly weakened amiloride block (8Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (240) Google Scholar), suggesting that the backbone assumes ϕ and ψ angles uniquely accessible to Gly at this position. In the only resolved structure of a protein with bound amiloride, that of urokinase-type plasminogen activator, several Ser and Gly residues were involved in the coordination of amiloride through either the backbone carbonyl or the Ser hydroxyl (20Zeslawska E. Schweinitz A. Karcher A. Sondermann P. Sperl S. Sturzebecher J. Jacob U. J. Mol. Biol. 2000; 301: 465-475Crossref PubMed Scopus (74) Google Scholar). Here, we have generated a series of point mutations at αSer-583 to explore the role of this residue in amiloride block. We found that residues containing rings have large effects on amiloride block, whereas substitutions that do not introduce rings have moderate or no effect on amiloride block. Interestingly, the presence of a side chain capable of forming H-bonds is not required for high affinity block. Furthermore, certain mutations altered the electric field of the conducting pore, a change that altered the gating behavior of ENaC. In addition, one of the mutations at this site generated a simple voltage-sensitive gate. These findings support a pore block model for amiloride inhibition of ENaC and give insight to the orientation of αSer-583 in the channel pore. Site-directed Mutagenesis—All ENaC clones used in this study are mouse ENaC subunits whose cDNAs were inserted into pBluescript SK (Stratagene, La Jolla, CA) (21Ahn Y.J. Brooker D.R. Kosari F. Harte B.J. Li J. Mackler S.A. Kleyman T.R. Am. J. Physiol. 1999; 277: F121-F129Crossref PubMed Google Scholar). Point mutations were generated by using a PCR-based method as previously described (22Sheng S. Perry C.J. Kleyman T.R. J. Biol. Chem. 2002; 277: 50098-50111Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). ENaC Expression—Stage V and VI oocytes free of follicle cell layers were injected with 1–4 ng of cRNA for each mouse ENaC subunit/oocyte and incubated at 18 °C for 24–72 h 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, 0.82 mm MgSO4, 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, 100 μg/ml gentamicin sulfate, pH 7.4). Where indicated, cRNAs encoding β and γ subunits truncated at Arg-564 and Arg-583, respectively, were injected instead of cRNAs encoding wild type β and γ subunits to increase functional expression (23Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (399) Google Scholar). Two-electrode Voltage Clamp—Two-electrode voltage clamp was performed using a DigiData 1320A interface and a GeneClamp 500B voltage clamp amplifier (Axon Instruments, Foster City, CA). Data acquisition and analyses were performed using pClamp 8.2 software (Axon Instruments) on a 1.5-GHz Pentium 4 PC (Gateway 2000 Inc., North Sioux City, S.D.). Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota, FL) with a Micropipette Puller (Sutter Instrument Co., Novato, CA) and had resistance of 0.5–5 megaohms when filled with 3 m KCl and inserted into the bath solution. Oocytes were maintained in a recording chamber (Automate Scientific, San Francisco, CA) with 20 μl of bath solution and continuously perfused with bath solution at a flow rate of 4–5 ml/min. The bath solution contained 110 mm NaCl, 2 mm KCl, 2 mm CaCl2, 10 mm HEPES, pH 7.4. All experiments were performed at ambient temperatures (20–24 °C). For experiments measuring apparent drug affinity, inhibitors were diluted from 0.5 m stock solutions in Me2SO into the bath solution and delivered to the oocyte by perfusion. Prior to measurement at each indicated inhibitor concentration, oocytes were perfused for 10 s at that inhibitor concentration. Amiloride Ki was determined by nonlinear curve fitting (Igor Pro 4.0.9.1, Oswego, OR) using Equation 1, IIo=KiKi+[Inh]+C (Eq. 1) where I is the Na+ current, Io is the Na+ current measured in the absence of inhibitor, [Inh] is the inhibitor concentration, and C is the inhibitor-insensitive component of the current. Current-voltage relationships were determined by clamping oocytes in a series of voltage steps (500 ms) from –140 to 60 mV in 20-mV increments, whereas whole cell currents were measured 400 ms after initiation of each voltage step. Oocytes were clamped at 0 mV for 50 ms between voltage steps. A tail protocol was also employed. Na+ currents were measured from oocytes clamped at Vhold for 2 s, followed by Vtest for 2 s. Vhold was 20 mV, and Vtest varied from –140 to 60 mV in 40-mV increments unless otherwise indicated. Itail and Iss were measured 50 and 1950 ms, respectively, after application of Vtest. Patch Clamp—Vitelline membranes of oocytes were removed manually following incubation of the oocytes at room temperature in modified Barth's saline supplemented with 200 mm sucrose. Oocytes were then transferred to a recording chamber with bath solution and allowed to recover for 10 min before clamping. The bath and pipette solutions contained 110 mm LiCl, 2 mm KCl, 2 mm CaCl2, 10 mm HEPES, pH 7.4. Glass pipettes with tip resistances of 5–20 megaohms were used. For single channel conductance measurements, single channel currents were recorded in excised inside-out configuration using an Axopatch 200B amplifier (Axon Instruments) and a DigiData 1322A interface (Axon Instruments) connected to a Pentium 4 PC (Gateway). Single channel recordings were acquired using pClamp 9.0 (Axon Instruments) at 20 kHz, filtered at 1000 Hz by a 4-pole low pass Bessel filter built in the amplifier, and stored on the hard disk. Single channel currents were further filtered at 100 Hz with a Gaussian filter for display and analysis. Transitions with duration Asn, Gln, Ser (wt) >Gly, Cys >Ile, Thr >Ala >Val >His >Phe >Tyr with wt-ENaC having a Ki of 130 ± 40 nm (Table I). Seven of the mutations had modest effects on apparent amiloride affinity, causing changes that were 16-fold or less. αS583V had a greater effect, weakening apparent affinity by 35-fold. Notably, αS583N and αS583Q did not significantly alter amiloride Ki, whereas αS583L increased apparent affinity by 4-fold. In contrast, all three ring residues had strong effects: αS583H weakened amiloride Ki by 230-fold, whereas both αS583F and αS583Y weakened amiloride Ki by more than three orders of magnitude.Table IEffect of αSer-583 mutation on amiloride and benzamil bindingα583nAmiloride Ki-100 mVδnBenzamil Ki-100 mVμmμmSer (wt)60.13 ± 0.040.19 ± 0.0350.0051 ± 0.0002Ala62.1 ± 0.3*0.18 ± 0.0250.28 ± 0.06*Cys60.6 ± 0.06*0.43 ± 0.03*60.12 ± 0.03*Phe8170 ± 10*0.24 ± 0.022054 ± 2*Gly60.6 ± 0.2*0.14 ± 0.0350.008 ± 0.002His530 ± 10*0.35 ± 0.03*546 ± 9*Ile141.56 ± 0.03*0.70 ± 0.03*80.16 ± 0.04*Leu110.031 ± 0.002*0.11 ± 0.02*60.0047 ± 0.0002Asn80.076 ± 0.0070.15 ± 0.0350.011 ± 0.001Gln50.088 ± 0.0020.22 ± 0.0350.026 ± 0.005*Thr61.6 ± 0.1*0.54 ± 0.03*50.04 ± 0.001*Val54.5 ± 0.9*0.71 ± 0.03*50.6 ± 0.1*Tyr8>300ND16171 ± 8* Open table in a new tab Parallel to experiments with amiloride, we also measured the apparent affinity of these mutants for benzamil, an amiloride derivative with a benzyl substitution off of the guanidinium moiety. Our results with benzamil are similar to those we found with amiloride (Fig. 1). The order of apparent benzamil affinity for these mutants was Leu, Ser (wt), Gly, Asn >Gln >Thr >Cys, Ile >Ala >Val >His, Phe >Tyr with wt-ENaC having a Ki value of 5.1 ± 0.2 nm (Table I). Five of the mutations had modest effects on apparent benzamil affinity, causing less than an 8-fold change. The Cys, Ile, Ala, and Val mutations had stronger effects, weakening benzamil Ki by 24-, 30-, 55-, and 120-fold, respectively. Dramatically, however, and paralleling our amiloride results, all three mutations containing ring structures weakened apparent benzamil binding by ∼four orders of magnitude or more. Interestingly, αS583Gβγ and wt-ENaC had similar apparent affinities for benzamil, which contrasts with αS583Gβγ having a weaker apparent affinity for amiloride as compared with wt-ENaC. This is because αS583Gβγ has a 75-fold greater apparent affinity for benzamil than amiloride, the largest such difference among all mutants. Effect of αSer-583 Mutations on Electrical Depth of Amiloride Binding—To further study the role of αSer-583 in the interaction between ENaC and amiloride, we examined the voltage dependence of amiloride block of the mutant channels. We measured amiloride Ki at 20-mV increments between –140 and –20 mm for each of the mutants studied here (Fig. 2). The voltage dependence of amiloride inhibition was then characterized by fitting the data in Fig. 2 to Woodhull's ionic block model, which provides an estimate for the electrical depth of the amiloride binding site within the channel (24Eyring H. Eyring E.M. Modern Chemical Kinetics. Van Nostrand Reinhold Company, New York1963Google Scholar, 25Woodhull A.M. J. Gen. Physiol. 1973; 61: 687-708Crossref PubMed Scopus (1232) Google Scholar). This model is derived from the application of Eyring rate theory to a simple scheme where the ionic blocker only accesses its binding site in the channel from the external side of the membrane, as shown in Equation 2. Ki(V)=Kioexp(zδFVRT) (Eq. 2) Here, Ki (V) is the amiloride Ki at a given electric potential, Kio is the amiloride Ki in the absence of an electric field, δ is the fraction of the membrane potential acting on the site, and z, F, V, R, and T are the valence of the blocking ion, the Faraday, the electrical potential across the membrane, the gas constant, and the absolute temperature, respectively. Five mutants, namely αS583A, αS583F, αS583G, αS583N, and αS583Q, had δ values that were not significantly different from that of wt-ENaC (Table I). Five other mutants, namely αS583H, αS583C, αS583T, αS583I, and αS583V, had δ values that were significantly higher than that of wt-ENaC. Interestingly, the three mutations that had the highest δ values, causing an average 3.4-fold increase versus wt-ENaC, were those with amino acid side chains that introduced a β-methyl group at α583 (i.e. Thr, Ile, and Val). Effect of αSer-583 Mutations on Current Rectification— Shown in Fig. 3 are the current-voltage (I-V) relationships for wt-ENaC and selected mutant ENaC channels in the absence of amiloride, measured 400 ms after application of the indicated voltages. As has been previously reported, wt-ENaC expressed in oocytes with a high internal [Na+] exhibits slight inward rectification and therefore has a near linear I-V relationship (26Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar). Five of the αSer-583 mutations, namely Leu, Gly, Asn, Gln, and Ala, behaved similarly to wt-ENaC (data not shown), whereas the remaining seven exhibited different I-V relationships. In the cases of αS583F and αS583Y, we observed outward rectification. The current-voltage relationship was simulated by characterizing the population of channels as having two distinct activity states with the voltage-dependent transition between the two states governed by a voltage-dependent Boltzmann distribution. This model is described by Equations 3 and 4 P(V)=(1+e−s(V−V1/2))−1 (Eq. 3) I=A·(V−VNa)·{P(V)+r−1[1−P(V)]} (Eq. 4) where P(V) is the probability of the channel being in the high activity state at a given voltage, V½ is the midpoint voltage of the transition, s is the slope of the S-shaped function at the inflection point, VNa is the reversal potential for Na+, r is the ratio between the high and low channel activity states, and A is a scaling factor so that I is –1 at –100 mV. Fits of this model to the data for αS583FβTγT and αS583YβTγT channels are shown in Fig. 3. Whole cell conductance was 1.6- and 2.7-fold higher at depolarizing versus hyperpolarizing clamping voltages for oocytes expressing αS583FβTγT and αS583YβTγT channels, respectively (Table II). For both mutants, V½ was similar to the reverse potential, suggesting that rectification is closely associated with the direction of the ion flux. Indeed, equivalent fits were obtained with one less variable by setting V1½ equal to VNa (fit not shown).Table IIFitted values for I–V plots of selected αSer-583 mutantsα583nV1/2VNarmVmVSer (wt)11-66 ± 73.3 ± 0.21.08 ± 0.01Thr11-88 ± 191.1 ± 1.42.7 ± 1Cys12-58 ± 45.1 ± 0.71.5 ± 0.1Ile22-65 ± 7-5 ± 22.6 ± 0.5Val10-120 ± 60-7 ± 43 ± 3Phe28-11 ± 18-2.7 ± 0.81.6 ± 0.2Tyr245 ± 8-2.5 ± 1.22.7 ± 0.1 Open table in a new tab In the case of oocytes expressing αS583Cβγ, αS583Iβγ, αS583Tβγ, or αS583Vβγ channels, we observed greater inward rectification relative to wt-ENaC. All four mutant channels were satisfactorily simulated by the model described above. The degree of rectification among three of these mutants was similar, having an average 2.8 ± 0.2-fold enhanced whole cell conductance at highly hyperpolarizing versus depolarizing potentials, with the rectification of αS583C being less pronounced with only a 1.5-fold change in activity. The V½ values were also similar among the four, having an average value of –83 ± 28 mV. Interestingly, three of these mutations are the only naturally occurring amino acids that have a methyl group on the β-carbon (see "Discussion"). Effect of αSer-583 Mutations on Single Channel Properties— Given that the time between current measurements during the I-V protocol was less than 1 s, we hypothesized that the rectification induced by these mutations was due to either a voltage-dependent conductance (g) or open probability (Po) effect and not due to changes in the number of channels in the membrane. To test this hypothesis, we performed single channel recordings of the αS583Tβγ channel (Fig. 4). In excised inside-out patches, in which both the bath and the pipette contained identical solutions with 110 mm Li+, we observed that the I-V relationship was linear from –120 to 60 mV (Fig. 4A). We found that the αS583T mutation reduced the single channel conductance with Li+ as the charge carrier, with g equal to 3.2 ± 0.1 pS, compared with wt-ENaC, which has a reported g value of 8.1 pS (27Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar). These data suggest that the inward rectification of αS583Tβγ is the result of a voltage-sensitive Po effect rather than a voltage-dependent g effect. To measure this effect directly, we measured the N·Po of αS583Tβγ in cell-attached patches, with 110 mm Li+ in the pipette solution (Fig. 4B). We observed that N·Po increased with hyperpolarization, such that from –40 to –80 mV the value of N·Po almost doubles. Assuming that N remains constant for each patch during the time of the experiment, these data demonstrate that the inward rectification of αS583Tβγ is the result of a voltage-dependent Po effect. We were unable to perform single channel experiments with the outwardly rectifying mutants αS583F and αS583Y, perhaps because of the low conductance evidenced by the necessity of coexpressing these mutants with βT and γT subunits to achieve functional expression. In lieu of single channel data, we performed a tail current protocol using two-electrode voltage clamp (Fig. 5). In these experiments, the membrane potential is held at a given voltage (Vhold, 20 mV) for a time sufficient to achieve steady state, after which the membrane potential is changed to a test value (Vtest). This is then repeated for a series of test potentials (Fig. 5, A and B). I-V relationships measured immediately upon change to Vtest (current defined as Itail) that deviate from linearity reflect either non-linear single channel conductance or very rapid Po changes. Changes in current that occur after Itail with a time constant on the order of seconds or faster can be attributed to changes in Po (Fig. 5C). Apparent changes in Po were then fit using the Boltzmann equation as shown in Equations 5 and 6 Po(V)=[1+exp(w−zgqeVkBT)]−1 (Eq. 5) ΔPo=Po(Vtest)−Po(Vhold)Po(Vhold)·100 (Eq. 6) where w is the conformational energy increase upon opening the channel in the absence of a membrane potential, zg is the gating charge, qe is the elemental charge, kB is Boltzmann's constant, and V and T are as described above. Values of w determine the Po in the absence of an electric field, whereas zg represents the net charge moved across the electric field during gating. Values of w determined by fitting had large confidence intervals (>95%); however, those of zg, were smaller. For the αS583T mutant, we observed a linear I-V relationship at Itail but an inwardly rectifying I-V relationship 2 s after application of the test potential (Iss), giving a zg value of –0.15 ± 0.02. These results clearly parallel the single channel results for αS583T in implicating a voltage-dependent Po change as responsible for the inward rectification of αS583T. Using the same rationale to analyze the other channels, we found that the Po of the wild type channel has only slight voltage dependence (zg = –0.04 ± 0.02). In contrast to wt-ENaC, the αS583C, αS583I, and αS583V mutants behave similarly to the αS583T mutant, giving zg values of –0.11 to –0.14. We note, however, that the I-V relationships taken at Itail for the αS583I and αS583V mutants do exhibit slight inward rectification. This is due, at least in part, to measuring Itail 50 ms after switching to Vtest in order to avoid current contamination from the capacitance current. However, we cannot exclude a non-linear single channel I-V relationship for these two mutants. Distinct
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