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Permeation of Large Tetra-alkylammonium Cations through Mutant and Wild-Type Voltage-gated Sodium Channels as Revealed by Relief of Block at High Voltage

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Permeation of Large Tetra-alkylammonium Cations through Mutant and Wild-Type Voltage-gated Sodium Channels as Revealed by Relief of Block at High Voltage
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   435   J. Gen. Physiol.  © The Rockefeller University Press •  0022-1295/2000/04/435/19 $5.00Volume 115April 2000435–453http://www.jgp.org/cgi/content/full/115/4/435   Permeation of Large Tetra-alkylammonium Cations through Mutant and Wild-Type Voltage-gated Sodium Channels as Revealed by Relief of Block at High Voltage  Chien-Jung Huang,* Isabelle Favre,* and  Edward Moczydlowski*   ‡  From the *Department of Pharmacology and ‡  Department of Cellular and Molecular Physiology, Yale University Medical School,New Haven, Connecticut 06520-8066   abstract   Many large organic cations are potent blockers of K      channels and other cation-selective channelsbelonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter andexit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in theDEKA locus of voltage-gated Na      channels is an important determinant of Na      /K      discrimination, exclusion of   Ca   2      , and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residueof the DEKA locus interacts with internal tetra-alkylammonium cations (TAA      ) that block Na      channels in a volt-age-dependent fashion. We investigated block by a series of TAA      cations of the wild-type rat muscle Na      channel(DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA    andlarger TAA      cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by   large TAA      cations as revealed by a positive inflection in the macroscopic I–V curve at voltages greater than    140mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) butnot small (e.g., TEA      ) symmetrical TAA      cations. The DEKA wild-type channel and the DERA mutant exhibit asimilar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a)hydrophobic TAA      cations with a molecular diameter as large as 15 Å can permeate Na      channels from inside tooutside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important de-terminant of inward rectification and internal block in Na      channels. From these observations, we suggest that hy-drophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may func-tion in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeationbehavior of large TAA      cations and potentially other kinds of local anesthetic molecules.   key words:   local anesthetic • ionic selectivity • Na      channel • selectivity filter • tetraethylammonium  INTRODUCTION  Tetra-alkylammonium (TAA     )   1  cations such as TEA     are known to inhibit ionic currents through channelproteins by transiently binding in the ion conductionpathway and blocking the flow of current (Tasaki andHagiwara, 1957; Armstrong and Binstock, 1965; Hille,1967). The fact that K     channels often differ in sensitiv-ity to block by internal and external TAA     s has led tothe picture that these channels have distinct internaland external binding sites for such molecules locatedin antechambers separated by a narrower tunnel in themiddle of the pore that cannot readily be penetratedby large organic cations (Armstrong, 1992;Hille, 1992).Structure–activity studies have identified certain aminoacid residues in K     channel proteins that influenceTEA     block (MacKinnon and Yellen, 1990; Kavanaughet al., 1991; Yellen et al., 1991; Heginbotham andMacKinnon, 1992; Choi et al., 1993). In the crystalstructure of a K     channel protein (KcsA), residues ho-mologous to those that determine the binding affinityof external and internal TEA     in related K     channelsare located precisely at the respective outer and innerentrances of the narrowest region of the pore calledthe selectivity filter (Doyle et al., 1998). In the structure of KcsA, this filter is a narrow tunnel that measures   3Å in diameter by 12 Å in length. Voltage-gated Na     and Ca   2     channels are structurallyrelated to K     channels as members of a homologous su-perfamily of cation-selective channels (Pongs et al.,1988; Jan and Jan, 1990). In particular, these three typesof channels share a structural motif called the P-regionlocated between the M1/M2 transmembrane helices ofKcsA or between the corresponding S5/S6 presumedhelices of voltage-gated K     , Na     , and Ca   2     channels(MacKinnon, 1995; Moczydlowski, 1998). In all three  Address correspondence to Edward Moczydlowski, Depart-ment of Pharmacology, Yale University Medical School, 333Cedar St., New Haven, CT 06520-8066. Fax: 203-785-7670;E-mail: edward.moczydlowski@yale.edu   1  Abbreviations used in this paper:      -CTX,   -conotoxin GIIIB; MA     ,methylammonium; STX, saxitoxin; TAA     , tetra-alkylammonium;TBA     , tetrabutylammonium; THexA     , tetrahexylammonium; TMA     ,tetramethylammonium; TPA     , tetrapropylammonium; TPeA     , tetra-pentylammonium; TTX, tetrodotoxin.  onN  ov  em b  er 1 4  ,2  0 1  3  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published April 1, 2000   436  Voltage-dependent Relief of Na      Channel Block by Large Organic Cations   types of channels, specific conserved residues in theP-region determine ionic selectivity (Heinemann et al.,1992; Yang et al., 1993; Heginbotham et al., 1994). Thestructure of KcsA revealed that K     selectivity in tet-rameric K     channels srcinates from a K     -binding re-gion consisting of four rings of carbonyl oxygen atomsof the peptide backbone contributed by residues of astrongly conserved signature sequence. However, manystudies indicate that the mechanism of ionic selectivityand the structure of the analogous selectivity filter arelikely to be quite different for Na     and Ca   2     channelsversus K     channels. Ionic selectivity in pseudotetramericNa     and Ca   2     channels is known to be intimately relatedto a set of mostly charged residues located near theouter mouth of the pore (Heinemann et al., 1992; Yanget al., 1993; Favre et al., 1996). These residues are calledthe EEEE locus in Ca   2     channels and the DEKA locus inNa     channels. The notation EEEE/DEKA correspondsto the single letter code for conserved amino acid resi-dues that can be readily aligned in homologous domainsI, II, III, and IV of Ca   2     /Na     channels, respectively. In apreceding study from our laboratory, the conserved Lysresidue in domain III of the DEKA locus of Na channels,K(III), was identified as a major determinant of the mo-lecular sieving behavior of the rat skeletal muscle Na-channel (    1) with respect to organic cations (Sun et al.,1997). Mutation of this Lys residue to Ala, as in the mu-tation of DEKA to DEAA, produces a channel that is per-meable to many organic cations ranging in size frommethylammonium (3.8-Å diameter) to TEA     (8.2-Å di-ameter), as demonstrated by direct measurement of in-ward currents carried by these cations. This latter behav-ior is quite different from native Na     channels, whichare effectively impermeable to external organic cationslarger than guanidinium, a fact that has been used to es-tablish 3.2   5.2 Å as the cutoff area of the narrowestcross-section of the native pore (Hille, 1971, 1972).The enhanced permeability of the DEAA mutant tolarge organic cations provides a unique opportunity toexplore the mechanism of block by TAA     cations andrelated local anesthetic drugs that are known to prefer-entially block voltage-gated Na     channels from the in-tracellular side. If the K(III) residue of the DEKA locuscorresponds to the location of a major energy barrierfor the movement of large cations through the pore,then lowering this energy barrier by mutation may en-hance the permeability of blocking cations that enterthe channel from the inside. Alternatively, if other sig-nificant energy barriers are located between the DEKAlocus and the intracellular pore entrance, then suchmutant Na     channels may exhibit asymmetric perme-ability to organic cations. On this basis, we hypothesizedthat removal of a structural element such as a Lys resi-due that limits conduction of organic cations may revealor enhance a phenomenon known as voltage-depen- dent relief of block (French and Wells, 1977; Frenchand Shoukimas, 1985), in which a large positive voltageapplied on the same side as a cationic blocker allowssuch blocking ions to permeate through the channel.To address the question of whether the K(III) residue ofthe DEKA locus corresponds to such an energy barrier,we studied internal block of a DEAA mutant Na     chan-nel by a series of symmetrical TAA     s ranging in sizefrom tetramethylammonium (TMA     ) to tetrahexylam-monium (THexA     ). We found that internal block ofoutward alkali cation current through the DEAA mu-tant by large TAA     cations such as tetrapentylammo-nium (TPeA     ) is indeed relieved in a steeply voltage-dependent fashion. Paradoxically, this effect is enhancedfor large versus small TAA     derivatives, implying that hy-drophobic interactions with long n   -alkyl groups actuallyfacilitate the voltage-driven permeation of very large or-ganic cations through this aqueous pore. In addition,we find that the wild-type Na     channel and a DERA mu-tant, in which the K(III) residue is replaced by Arg, alsoexhibit voltage-dependent relief of block by large chainTAA     s, but with apparently different efficiency. The re-sults show that a positively charged residue at the K(III)position of the DEKA locus is not an absolute structuralimpediment to the movement of large cations throughthe pore. Implications of these findings for the interpre-tation of molecular sieving studies of Na     channels andblocking interactions of TAA     cations are discussed.  MATERIALS AND METHODS  Expression of Wild-Type and Mutant Na      Channels    Na      channels studied in this paper are the wild-type    1 rat skele-tal muscle isoform (Trimmer et al., 1989) and two mutants of thisclone that contain substitutions, K1237A or K1237R. The wild-type and the latter two mutants are referred to, respectively, asDEKA, DEAA, and DERA according to the single letter code forresidues in domains I–IV of the DEKA locus, a conserved regionin Na      channels that aligns with the EEEE locus of Ca   2      channels(Heinemann et al., 1992; Sun et al., 1997). DEKA and DEAAclones were studied in stably transfected human fibroblastHEK293 cells. The DERA clone was studied in HEK293 cells thatwere transiently transfected with a DERA/pcDNA3 vector usingEffectene reagent (QIAGEN Inc.). Construction of the muta-tions, subcloning into pcDNA3 expression vector, and othermethods for transfection, generation, and propagation of stablecell lines have been described in previous publications (Favre etal., 1996; Sun et al., 1997).  Solutions and Electrophysiology    The standard extracellular Na      bath solution was (mM) 140NaCl, 3 KCl, 2 MgCl   2   , 2 CaCl   2   , 10 glucose, and 10 HEPES-NaOH,pH 7.3. The standard intracellular Cs      /Na      pipette solution was(mM) 125 CsF, 2 MgCl   2   , 1.1 EGTA, 10 glucose, 20 Na      -HEPES,pH 7.3. Intracellular pipette solutions containing tetrapropyl-ammonium (TPA      ), tetrabutylammonium (TBA      ), TPeA      ,THexA      , and QX-314      were prepared by adding these blockersto standard Cs      /Na      pipette solution at the desired concentra-tion. The composition of other solutions for testing permeability   onN  ov  em b  er 1 4  ,2  0 1  3  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published April 1, 2000   437  Huang et al.   to external TEA      , TMA      , MA      , and Ca   2      or block by internalTEA      are given in the figure legends.Patch-clamp electrodes were constructed with a commercial pi-pette puller using Kimax 50 borosilicate capillary glass (Fisher Sci-entific Co.) without additional fire polishing. The measured pi-pette resistance was 1–2 M      when filled with standard Cs      /Na      pipette solution. Whole-cell voltage-clamp recording was per-formed at room temperature (     22      C) using an amplifier (EPC-9;HEKA Electronik) with Pulse and Pulse-fit software (InstrutechCorp.). Stably transfected HEK293 cells were seeded for growthon glass cover slips and used for electrophysiological recordingwithin 12–36 h. Transiently transfected cells expressing the DERAmutant channel were used for recording 48 h after transfection.The peak inward current in standard Na      bath solution and Cs      /Na      pipette solution of typical cells selected for recording DEKA,DEAA, and DERA current was    4.3  0.4 nA (mean  SEM,   n    6),    4.1  0.7 nA (   n     12), and    1.6  0.4 nA (   n     5), respec-tively. Cancellation of residual capacitance transients and linearleak subtraction was carried out using a programmed P/4 nega-tive pulse protocol delivered at    120 mV. The series resistancecompensation function of the amplifier was routinely used at 74–79% compensation to minimize voltage error. Nevertheless, someexperiments for the DEAA mutant (e.g., see Figs. 2 and 5) in-volved measurements of outward currents as large as 50–100 nA atthe highest positive voltage (  200 mV). In such cases, the effect ofvoltage errors on our analysis and interpretation was minimizedby discarding data sets from cells that exhibited  50 nA of out-ward current at  200 mV. In addition, the effect of voltage errordue to series resistance, R  S , was estimated for the data of Figs. 2and 5 by correcting the applied voltage by the voltage drop acrossthe noncompensated fraction, f  N , of measured R  S (2.3  0.2 M  , n     5), using Ohm’s Law,  V  If  N R  S  (Marty and Neher, 1995).Current–voltage data were collected by recording responses toa consecutive series of step voltage pulses increasing by  10 mVfrom a holding potential of  120 to  200 mV with a pulse dura-tion of 10 ms. The interval between consecutive voltage pulseswas 0.5 or 1 s as noted, except in experiments where it was variedfrom 0.5 to 10 s for investigation of use-dependent block. Datacollection was begun 10 min after whole cell break-in when in-ward Na   current reached a relatively stable level after intracellu-lar perfusion. Data were collected during continuous gravity-fedperfusion of the extracellular bath solution using a commercialpatch perfusion chamber (Warner Instrument Co.).The permeability of extracellular TEA   and MA   was investi-gated by measuring the change in reversal potential,  V R , uponchanging the extracellular solution from the standard Na   bathsolution to a different solution containing the cation of interest(e.g., see Figs. 1 A and 7). Permeability ratios of these extracellu-lar cations relative to Na   were calculated by the method of Hille(1971) using  V R  values corrected for junction potentials andequations described previously (Sun et al., 1997). Data Analysis and Modeling  Peak current values were measured and plotted as a function ofthe pulse voltage. In compiling the results, peak I-V data was of-ten normalized by dividing the measured current by the maxi-mum inward current of a given cell. Such data sets collectedfrom three to eight cells were then averaged and plotted as themean normalized current with error bars given (  SEM).In Fig. 2 B (below), current values for 10 and 50  M internalTPeA   were normalized by dividing each point by the expectedpeak inward current in the absence of internal TPeA  . The mag-nitude of the unblocked current was estimated by fitting datapoints at voltages less than  80 mV to a simple model of voltage-dependent block as described by Eqs. 1 and 2 with outward per-meation of the blocker disregarded by setting k   2  equal to 0. Thisprocedure yields a reasonable estimate of the maximal conduc-tance, G  max , in the absence of blocker. This estimate of G  max  isthen used to generate an I-V relation for the expected unblockedcurrent by setting the blocker concentration, [B in ], equal to zeroin Eq. 1. The absolute value of the maximal expected inward cur-rent is then used to normalize the measured current in the pres-ence of internal TPeA   to produce a macroscopic I-V curve thatis appropriately scaled with respect to that expected in the ab-sence of an internal blocker. This procedure is mathematicallysimilar to that used in Figure 5 of O’Leary and Horn (1994),which describes the blocking effect of internal TEA  , TPA  , andTBA   on the human heart Na   channel as measured by whole-cell recording.Nonlinear regression fitting of peak I-V data to the model ofScheme I using Eqs. 1 and 2 was performed using the Marquardt-Levenberg algorithm as part of the Sigmaplot 4.0 software pack-age (SPSS Inc.). Materials  Chloride salts of TAA   cations were purchased from AldrichChemical Co. (TPA  , TBA  , TPeA  ) or Sigma Chemical Co.(TEA  , THexA  ). Saxitoxin and tetrodotoxin were purchasedfrom Calbiochem Corp.  -Conotoxin GIIIB was obtained fromBachem. The quaternary lidocaine derivative QX-314   was ob-tained from Alomone Laboratories. RESULTS Voltage-gated Na   channels are preferentially blockedby many different hydrophobic organic cations fromthe intracellular side. For example, QX-314  , the qua-ternary ammonium derivative of lidocaine, a local anes-thetic, readily blocks Na   channels when present onthe intracellular side of axons, cells, or planar bilayers(Strichartz, 1973; Moczydlowski et al., 1986; Wang,1988; Gingrich et al., 1993; Zamponi et al., 1993). Vari-ous measurements of the steady state voltage depen-dence of block of open Na   channels by QX-314  ,TAA   derivatives, and related molecules, indicate thatthere is an internal site (or sites) for organic cationsthat senses 40–70% of the applied voltage from the in-side of the membrane (Yamamoto and Yeh, 1984; Wanget al., 1991; Gingrich et al., 1993; French et al., 1998).At the opposite side of Na   channels, there is anotherblocking site for guanidinium toxins such as tetrodo-toxin (TTX) and saxitoxin (STX). Amino acid residuesthat determine the affinity for TTX/STX binding arelocated at or close to the COOH-terminal side of resi-dues that comprise the DEKA locus, an apparent ring-like structure of mostly charged residues that controlthe relative permeability to Na  , K  , and Ca 2   (Terlauet al., 1991; Heinemann et al., 1992). The D, E, and K,residues of the DEKA locus also determine molecularsieving properties of the  1 Na   channel as monitoredby inward current carried by various organic cations inthe extracellular solution (Sun et al., 1997). This sug-gests that the DEKA locus forms a constricted regionthat separates the toxin binding site on the outside   onN  ov  em b  er 1 4  ,2  0 1  3  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published April 1, 2000  438 Voltage-dependent Relief of Na    Channel Block by Large Organic Cations  from the blocking site for TAA   cations on the inside.If the DEKA locus forms such a constriction in struc-tural terms, or is the major energy barrier in kineticterms, then one might expect that internal organic cat-ions would become permeable or lose their blockingefficiency in a mutant such as DEAA that displays en-hanced permeation of organic cations from the out-side. To test this idea, we first examined whether inter-nal organic cations can carry outward currents in theDEAA mutant expressed in HEK293 cells. Asymmetry of Organic Cation Permeation through the DEAA Mutant Na    Channel  The control experiment of Fig. 1 shows that replace-ment of a standard extracellular Na   solution with a so-lution containing TEA   as the major external cation re-sults in a small inward current and a negative shift ofthe reversal potential of whole-cell peak current for theDEAA mutant. The magnitude of this shift (  24.6  0.3 mV) corresponds to a permeability ratio ofP TEA (out)/P Na    0.37  0.01 ( n     4), confirming thatexternal TEA   can permeate through this channel.TEA   is the largest cation that has thus far been foundto exhibit measurable inward current for the DEAAmutant (Sun et al., 1997). When the standard intracel-lular solution containing 125 mM Cs   plus 20 mM Na  was replaced with a pipette solution containing 115mM TEA-Cl, outward current could not be satisfactorilyresolved since it was  0.1 nA at  200 mV. This observa-tion suggests that the DEAA channel is essentially im-permeable to internal TEA  . To improve the possibilityof observing outward current carried by an organic cat-ion, we tested TMA  , a smaller TAA   derivative. Fig. 1B shows typical currents and peak I-V data obtained forcells recorded in the presence of 115 mM internalTMA   and 145 mM external Na  . This experiment suc-cessfully resolved a small outward current carried byTMA  , but strong rectification observed in the positivevoltage range implies that the conductance supportedby internal TMA   is small in comparison with externalNa  . The nominal reversal potential in this experimentcorresponds to a permeability ratio of P TMA (in)/P Na  of0.07, or at least sevenfold less 2  than the value ofP TMA (out)/P Na    0.50 that was previously measuredwith TMA   on the outside of the DEAA mutant (Sun etal., 1997). These results indicate that permeation ofsmall TAA   cations through the DEAA mutant is asym-metric in nature. TMA   carries current through thischannel in both directions, but both TMA   and TEA  are significantly more permeable when tested in the in-ward versus the outward direction. Relief of Block by Internal TPeA   at High Positive Voltage in DEAA Mutant and Wild-Type Na    Channels  The next question we addressed was whether block ofoutward alkali cation current by large hydrophobicTAA  s is altered in the DEAA mutant compared withwild type. Internal TPeA   has been previously character-ized as a potent blocker of outward Na   current for thewild-type human heart Na   channel (O’Leary et al.,1994; O’Leary and Horn, 1994). In this latter study,block was described by voltage-dependent binding ofTPeA   to a site located at an apparent electrical distanceof 0.41 from the inside with a K  d  of 9.8  M at 0 mV. Fig.2 shows typical currents and peak I-V data for HEK293cells expressing the DEAA mutant. Current records ob-tained under control conditions with 120 mM Cs   plus20 mM Na   in the internal solution were compared withsimilar recordings with 1, 10, or 50  M TPeA   added tothe pipette solution. The control records show thatlarge rapidly inactivating outward currents are observedunder these conditions. The outward current is a mix-ture of Cs   and Na   current since this mutant is rathernonselective for alkali cations with P Cs /P Na    0.57 forCs   tested on the outside (Sun et al., 1997). The controlpeak current in the DEAA mutant exhibits nearly ohmicbehavior in the positive voltage range up to  200 mV(Fig. 2 B, control). Fig. 2 B shows that addition of 10 or50  M TPeA   to the internal solution strongly sup-presses outward current carried by alkali cations. In thepositive voltage range up to about  140 mV, TPeA   be-haves as a voltage-dependent blocker as reported for theheart Na   channel (O’Leary et al., 1994).However, at voltages greater than  140 mV, there is asharp upturn of the peak I-V relationship with 10 and50  M TPeA   (Fig. 2 B). Inspection of the correspond-ing current traces (Fig. 2 A) shows that outward currentsrecorded with 10 and 50  M TPeA   display typicalrapid activation and inactivation kinetics of voltage-gated Na   channels. A positive inflection in the peakI-V relations of Fig. 2 B arises from a rather abrupt in-crease in transient current at voltages greater than  140 mV (Fig. 2 A). This kind of behavior is consistentwith voltage-dependent relief of TPeA  -blocked Na  channels, rather than an artifact due to activation of 2 This estimate of 0.07 for P TMA (in)/P Na  was calculated using the ap-parent reversal potential and an appropriate form of the Goldmann-Hodgkin-Katz voltage reversal equation for major monovalent cat-ions. However, this calculation does not take into account the changein junction potential that occurs in whole-cell recording after diffu-sional equilibration between the pipette contents and cell interior.As described by Marty and Neher (1995), the actual membrane po-tential will be more positive than the amplifier reading when thedominant cation (TMA  ) in the pipette solution is less mobile thanthe dominant anion (Cl  ). This means that the true reversal poten-tial in this experiment is more positive than the measured value andthat our estimate of the relative permeability of TMA  (in) is an up-per limit. Thus, this uncertainty does not compromise the conclusionthat internal TMA   has lower permeability than external TMA  .   onN  ov  em b  er 1 4  ,2  0 1  3  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published April 1, 2000  439 Huang et al. some other type of channel with different kinetics inHEK293 cells. Visual comparison of the time course ofthe outward current in the absence and presence ofTPeA   shows that the apparent rate of inactivation isdistinctly faster with TPeA   (Fig. 2 A). This phenome-non was also observed by O’Leary et al. (1994) andO’Leary and Horn (1994), who concluded that the fastcomponent of inactivation in the presence of internalTPeA   is related to the rate of association of TPeA   tothe open channel. Such changes in the kinetics of Na  currents by TAA   cations are interesting but beyondthe scope of the present study. Here we focus on themechanism of relief of block as assayed by the magni-tude of peak current.Because the absolute outward current in HEK293cells expressing the DEAA mutant can be as large as 100nA, we considered whether residual series resistance er-ror might lead to distortions in the measured peak I-Vrelations that could affect our interpretation. Uncom-pensated series resistance leads to distortions in the cur-rent time course and a failure to maintain the desiredclamp voltage (Armstrong and Gilly, 1992; Marty andNeher, 1995). This voltage error is proportionallygreater, the greater the current. In Figs. 2 B and 5 B, weshow the probable effect of this error on the measuredpeak I-V relations by correcting the applied voltage bythe estimated voltage drop due to uncompensated se-ries resistance. The greatest effect of this error is an un-derestimation of the actual magnitude of the peak cur-rent at high positive voltages. Since absolute current re-corded in the presence of blockers is substantiallyreduced relative to control, this error actually results ina greater distortion of the control records withoutblockers than the records taken in the presence ofTPeA  . Also, this error leads to an underestimate of thetrue slope of the peak I-V relation in the high voltage Figure  1.Permeation of external TEA   and internal TMA   as observed for the DEAA mutant. Whole-cell currents were recorded with aseries of consecutive 10-ms voltage steps of  10 mV from a holding potential of  120 mV at intervals of 1 s. (A) External TEA   carries asmall inward current. (Top) Typical records with standard Cs  /Na   pipette solution and standard Na   bath solution before (left) and af-ter (right) perfusion with TEA   bath solution (143 mM TEA-Cl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM glucose, 10 mM HEPES-TEA, pH 7.3).(Bottom) Normalized peak I-V curves (mean of four cells  SEM) in control Na   bath solution, after replacement with TEA   bath solu-tion, and again after washout of TEA   with Na   solution. (B) Internal TMA   carries a small outward current. (Top) Typical currents withTMA   pipette solution (100 mM TMA-Cl, 2 mM MgCl 2 , 1.5 mM EGTA, 10 mM glucose, 20 mM HEPES-TMA, pH 7.3) and standard Na  bath solution. Peak-normalized I-V data are shown at the bottom (mean of three cells  SEM). Solid lines simply connect I-V data points inFigs. 1–8 and do not signify any particular theory.   onN  ov  em b  er 1 4  ,2  0 1  3  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published April 1, 2000
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