Journal of Physiology (1997), 499.2, pp.361-367

Pore mutations in ShakerK+ channels distinguish between thesites of tetraethylammonium blockade and C-type inactivation

A. Molina, A. G. Castellano and J. Lopez-Barneo *

Departamento de Fisiologia Medica y Biofisica, Facultad de Medicina,Universidad de Sevilla, E-41009 Sevilla, Spain

1. We have studied the effect of external K+ and tetraethylammonium (TEA) on severalmutants of Shaker B K+ channels with amino acid substitutions in the pore which alter TEAaffinity and the rate of C-type inactivation. In all channels studied high external K+ makesC-type inactivation slower.

2. In the wild-type channel, TEA blockade is voltage dependent and produces slowing of theinactivation time course. However, in the double mutant channel (T449Y,D447E) TEAblockade, although of higher affinity, is voltage independent and does not affect the rate ofC-type inactivation.

3. Mutants with a charged amino acid at position 449 (T449K and T449E) are resistant to TEAblock. In these channels, C-type inactivation is also unaffected by TEA.

4. These results indicate that the sites where TEA blocks and competes with C-typeinactivation can be segregated. To modulate inactivation, TEA must enter deeply into thechannel mouth. These results suggest that C-type inactivation is not due to a large molecularrearrangement in the outer channel vestibule, but it is essentially produced by aconformational change restricted to a local site in the pore.

Inactivation is a fundamental property of many voltage-dependent K+ channels that determines important featuresof electrical signalling in excitable cells, such as the firingfrequency and the shape of individual action potentials (seeHille, 1992). In Shaker K+ channels there are two differentmechanisms of inactivation, referred to as N- and C-typeinactivation, which are associated with separate domains ofthe molecule. N-type inactivation involves a region near theamino terminus which forms a tethered inactivating peptidethat can bind to a receptor site and occlude the internalmouth of the channel (Hoshi, Zagotta & Aldrich, 1990,1991). C-type inactivation is associated with a differentconformational change involving amino acids located in theouter channel mouth and in the S6 transmembrane region(Busch, Hurst, North, Adelman & Kavanaugh, 1991; Choi,Aldrich & Yellen, 1991; Hoshi et al. 1991; De Biasi, Hartman,Drewe, Taglialatela, Brown & Kirsch, 1993; Lopez-Barneo,Hoshi, Heinemann & Aldrich, 1993). Although in Shaker Bchannels the two inactivating processes coexist, in K+channels without the inactivating N-terminal peptide theC-type is possibly the major mechanism of inactivation.C-type inactivation can be slow, with time constants in therange of seconds, or very fast, occurring in a few milliseconds(Hoshi et al. 1991; Lopez-Barneo et al. 1993).

The molecular mechanism of C-type inactivation is unknown.C-type inactivation is inhibited by external tetraethyl-ammonium (TEA) (Grissmer & Cahalan, 1989; Choi et al.1991), by external K+ and, with less potency, by othermonovalent cations (L6pez-Barneo et al. 1993). It is alsoaltered by amino acid mutations in the pore. Single aminoacid substitutions in the position 449 of the Shaker K+channel lead to changes of three orders of magnitude in therate of C-type inactivation. They also alter the permeabilityproperties of the Shaker K+ channels in parallel with theeffects on C-type inactivation: those channels with a largerconductance tend to inactivate more slowly (Lopez-Barneoet al. 1993). These observations have been explained by a'foot-in-the-door' model of gating, in which external cations,or TEA, compete with C-type inactivation by occupancy ofa site in the pore that must be emptied before inactivationcan proceed (Lopez-Barneo et al. 1993; see also Baukrowitz& Yellen, 1996). However, the location of the site(s) wherethe cations compete with inactivation, as well as themolecular rearrangements underlying C-type inactivation,are not well defined. In tetrameric K+ channels the foura-subunits seem to participate co-operatively in C-typeinactivation (Ogielska, Zagotta, Hoshi, Heinemann, Haab &Aldrich, 1995; Panyi, Sheng, Tu & Deutsch, 1995). Recent

* To whom correspondence should be addressed.

This manuscript was accepted as a Short Paper for rapid publication.

6365 361

A. Molina, A. C. Castellano and J Lopez-Barneo

studies have suggested that during C-type inactivation thefour subunits come to closer apposition and that this motionresults in the exposure of several side-chains of amino acidslocated in the outer mouth of the channel (Liu, Jurman &Yellen, 1996). Externally applied TEA blocks K+ channels bysimultaneous interaction with several subunits (Heginbotham& MacKinnon, 1992; Kavanaugh, Hurst, Yakel, Varnum,Adelman & North, 1992; Ogielska et al. 1995), and thus TEAblockade and competition with C-type inactivation werebelieved to reflect the same molecular process. We haveconstructed several mutants of Shaker B K+ channels withamino acid substitutions in the pore that alter TEA affinityand C-type inactivation kinetics. The study of thesemutants indicates that the sites of TEA blockade andcompetition with C-type inactivation can be segregated.TEA appears to alter C-type inactivation only when itenters deeply into the channel mouth. These results suggestthat the conformational change in the pore causing C-typeinactivation is restricted to a local site, without directinvolvement of residues located in superficial regions of theouter channel mouth.

METHODSWe used as the wild-type construct the Shaker B A6-46 channelwhich has a deletion in the amino terminal that completely removesN-type inactivation (Hoshi et al. 1990). Point mutations were madeon this construct using mismatched oligonucleotides and thepolymerase chain reaction (PCR). Each of the 449 mutants weremade as described previously (L6pez-Barneo et al. 1993). We usedthe same methodology to introduce a glutamate in position 447 ofthe wild-type (T449T) or T449Y channels to obtain the mutantsD447E and T449Y,D449E. The cDNAs encoding for the variousmutant channels were subeloned in plasmid p513, a derivative ofpSG5 (Stratagene Ltd, Cambridge, UK). For all the mutations, theentire region of the PCR-amplified fragments was sequenced tocheck for the mutation and ensure against mistakes in the PCR.Chinese hamster ovary cells were transiently transfected with2-4 ,ug of the cDNAs by electroporation (Gene Pulser, Bio-RadLaboratories, Hercules, CA, USA). For transfection, cells wereresuspended in a sucrose-phosphate buffer and placed in cuvettesat room temperature. Electroporation parameters (350 V and125 ,uF) yielded typical time constant values of 24-26 ms. Afterelectroporation the cells were resuspended in culture medium(McCoy's 5A with supplements; BioWhittaker, Walkersville, MD,USA), plated on slivers of coverslips, and maintained in a CO2incubator at 37 °C until use.

Cells were used for electrophysiological recording 24-72 h afterplating. For the experiments, a coverslip was transferred to a-0-2 ml recording chamber and the cells were bathed with acontinuous flow of solution that could be completely replaced in lessthan 40 s. Potassium currents were recorded using the whole-cellconfiguration of the patch-clamp technique as adapted in ourlaboratory. We considered in the analysis only those cells withmacroscopic potassium currents between 0 5 and 3 nA at 20 mV;cells expressing larger or smaller currents were discarded. We usedlow resistance electrodes (1-3 MQ), capacity compensation andsubtraction of linear leakage and capacity currents. Seriesresistance compensation was not systematically used. The C-typeinactivation rate was estimated by fitting the inactivation timecourse with a single exponential function. Since in all the mutants

studied activation is much faster than inactivation, C-typeinactivation time courses could be compared by scaling theamplitudes of the whole-cell currents. The composition of thestandard external solution was (mM): 140 NaCl, 2 7 KCl, 2 5 CaCl2,4 MgCl2, 10 Hepes, pH 7*4. The composition of the standardsolution in the pipette and inside the cell was (mM): 80 KCl, 30potassium glutamate, 20 potassium fluoride, 4 MgATP, 10 Hepes,10 EGTA, pH 7 2. In the high K+ external solutions, 70 mM NaClwas replaced by 70 mm KCl. When external TEA was tested, weadded equimolar TEACI to replace NaCl. The holding potentialwas -80 mV. Experiments were performed at room temperature(,22 °C).

RESULTSFigure IA indicates the location of the amino acids mutatedin our study (447 and 449) using a model that illustrates thetransmembrane topology of the a-subunit of voltage-gatedK+ channels. The residue at position 449 varies amongcloned K+ channels (see Stuihmer et al. 1989) and determinesimportant functional properties, such as TEA sensitivity(MacKinnon & Yellen, 1990) and C-type inactivation rate(Lopez-Barneo et al. 1993). Aspartate at position 447 isimportant for cation selectivity (see Kirsch, Pascual & Shieh,1995). Our experiments aimed to distinguish between thesites of TEA blockade and C-type inactivation and werebased on previous work on a mutant channel (T449Y) whichhas tyrosine instead of threonine at position 449 and ishighly sensitive to TEA (MacKinnon & Yellen, 1990). TheA6-46 T449Y channel inactivates very slowly (timeconstant, -.-28 s) with a time course unaffected by externalK+ (Lopez-Barneo et al. 1993) or TEA (not shown). Whereasin the wild-type channel (T449T) TEA blockade is voltagedependent because it enters the pore and senses thetransmembrane electric field, in the mutant T449Y TEAblockade is voltage independent, which suggests that itbinds to a more superficial location in the ion conductingpathway (Heginbotham & MacKinnon, 1992). Because theT449Y channels inactivate too slowly to be compareddirectly with the wild-type channels, we designed a doublemutant with relatively fast C-type inactivation and highsensitivity to TEA. In the single mutant D447E,inactivation is much faster than in the wild-type channel(see below) and is also slowed by external K+ (authors'unpublished results); thus we introduced this mutation inthe T449Y channel to obtain the mutant T449Y, D447E.The basic properties of C-type inactivation of the wild-type(Shaker B A6-46; T449T) and the double mutant(T449Y,D447E) are summarized in Fig. lB and C, whichshows recordings of macroscopic currents. In the twochannel types the inactivation kinetics are exponential withsimilar time constant values (see Fig. 1 legend). Furthermore,in the two channel constructs, the C-type inactivation ratewas slowed down to the same extent by high external K+(Fig. lB and C and figure legend). Therefore, C-typeinactivation of the T449T and T449Y,D447E channelsshare similar properties: almost identical kinetics andmodulation by external K+.

J Physiol.499.2362

Pore mutants, TEA block and C-type inactivation

External K+ appears to prevent inactivation by occupancyof a site in the pore that must be emptied beforeinactivation can proceed (Lopez-Barneo et al. 1993) and asimilar effect is produced by a fast channel blocker such asTEA. The interaction of TEA with the C-inactivationprocess in the wild-type and mutant channels is illustratedin Fig. 2. Figure 2A a shows superimposed recordingsobtained from cells transfected with T449T channels,during long-lasting depolarizing pulses in standard ionicconditions (Control) and after addition of 30 mm TEA to theexternal solution. This TEA concentration, close to the EC50(MacKinnon & Yellen, 1990), decreased current amplitude

A

Extracellular

B T449T

a

to almost 50 %. Scaling of the two current traces (Fig. 2A b)illustrates that, as described previously (Grissmer &Cahalan, 1989; Choi et al. 1991), TEA produced an almost5-fold reduction in the rate of C-type inactivation (see Fig. 2legend). The effect of TEA on the T449T channel type wasclearly voltage dependent: for example, with 30 mm TEA,inhibition of current amplitude at a membrane potential of0 mV was 50 + 5% (n = 9) of control, but it was only of60 + 3% (n = 9) at +50 mV (means + S.D.). This indicatesthat in the process of blockade TEA enters into the poreand, hence, senses the transmembrane electric field. Inconditions similar to ours, it was previously deduced that

Pore

49

44

I Control

\70 mM K+

1 s i s

C T449Y,D447E

a b

Control

70 mM K+

1 s 1 s

Figure 1. Characteristics of C-type inactivation in wild-type (T449T) and in double mutant(T449Y, D447E) channelsA, proposed secondary structure of K+ channel a-subunits with the position of amino acids 447 and 449indicated. B, currents recorded in cells transfected with the wild-type T449T channels. Ba, current tracesrecorded with standard solutions (Control) and in the presence of 70 mm external K+. Bb, scaling of thecurrent traces to illustrate the reduction of C-type inactivation rate by external K+. The inactivation timeconstant of the T449T channel was 1 2 + 0-16 s (mean + S.D., n = 6) in control solution and increased to2-1 + 027 s (n = 5) in high external K+. C, currents recorded in cells transfected with the T449Y,D447Emutant channels using experimental protocols similar to those illustrated in in B. The inactivation timeconstant of the T449Y,D447E channel was I - + 0 11 s (n = 5) in control solution and increased to2-2 + 0'19 s (n = 5) in high external K+.

J Physiol. 499.2 363

364 A. Molina, A. C. Castei

external TEA moves into the pore to a depth which is about20% of the distance of the electric field (Heginbotham &MacKinnon, 1992). The effect of replacing aspartate atposition 447 by glutamate (D447E) is represented in Fig. 2B.Inactivation of this mutant in the control solution was-500-fold faster than that of the wild-type channel andwas also markedly decelerated by external TEA (see Fig. 2

A T449T

a

B D447E

a A

llano and J L6pez-Barneo

legend). As in other channel types with fast C-typeinactivation, TEA increased the amplitude of themacroscopic current, possibly because it competes withclosed-state inactivation and increases the number ofchannels that open on depolarization (see L6pez-Barneo et al.1993). Finally, the effect of TEA on the T449Y,D447Emutant is illustrated in Fig. 2C. The double mutation

b

Control

30 mM TEA nA

30 mM TEA

2 s

b

i30 mM TEA

1 nA

5 ms

C T449Y,D447E

a A b

10 mM TEA

nA

10 mM TEA

1 s

Figure 2. Interaction of external TEA with the wild-type (T449T) and the mutant channelsD447E and T449Y, D447EA, currents recorded in cells transfected with the T449T channels. A a, blockade of the current by 30 mMTEA; A b, scaling of the currents to illustrate the reduction in the C-type inactivation rate caused by TEA(time constant = 5 1 + 0X41 s; n = 4). Pulses to 0 mV were applied. B, currents recorded in cellstransfected with the D447E channel. Ba, increase in macroscopic current amplitude induced by 30 mmTEA; Bb, scaling of the currents to illustrate the reduction in the C-type inactivation rate caused by TEA.Pulses to 0 mV. The inactivation time constant was 2-6 + 0 7 ms (n = 14) in the control solution andincreased to 9-8 + 1-4 ms (n = 6) in 30 mm external TEA. C, currents recorded in cells transfected withthe T449Y,D447E channels. Ca, current traces recorded with the standard solution (Control) and in thepresence of 10 mm TEA. Cb, scaling of current traces indicating the insensitivity of C-type inactivation toTEA. The inactivation time constant with 10 mm TEA was 1-4 + 0 13 s (n = 4). Pulses to 0 mV.

J Physiol. 499.2

Pore mutants, TEA block and C-type inactivation

produced a -4-fold increase in the affinity of the channel forTEA (EC50 z 8 mM). Exposure of the double mutantchannels to 10 mm external TEA reduced the currentamplitude by more than half the control value (Fig. 2Ca) butdid not produce appreciable changes in the C-typeinactivation time course (Fig. 2Cb; see also Fig. 2 legend).TEA blockade of this channel variant was essentiallyvoltage independent: for example, with 10 mm TEA,inhibition of current amplitude at membrane potentials of 0and +50 mV was, respectively, 42 + 6% (n = 7) and44 + 4% (n = 7) of control. This indicates that althoughTEA blocked the channels with higher affinity (possibly dueto binding to the tyrosines at position 449; see Heginbotham& MacKinnon, 1992) it remained outside the transmembraneelectric field. Hence, the results demonstrate that TEA blockcan be segregated from C-type inactivation. Furthermore,they strongly suggest that TEA competes with C-typeinactivation only when it penetrates deeply into the pore.

A corollary of this proposal is that channels highly resistantto TEA block should have C-type inactivation ratesinsensitive to this agent. We tested this idea with a mutant

A T449K

a a

I Control

containing a positively charged amino acid (lysine) atlocation 449 (T449K), which is known to be resistant toTEA block (McKinnon & Yellen, 1990; Pardo et al. 1992)and to have fast C-type inactivation (Lopez-Barneo et al.1993). Figure 3A a shows that, as with other channelvariants, C-type inactivation of the T449K channels wasalso slowed by high external K+. By contrast, a highconcentration of TEA (70 mM) had no effect on either theamplitude or the C-type inactivation time course of theT449K macroscopic currents (Fig. 3A b and figure legend). Ithas been reported before that the introduction of glutamate(a negatively charged amino acid) at position 449 results inchannels which have C-type inactivation kineticscomparable to those of T449K channels and which are alsomodulated by external K+ (Lopez-Barneo et al. 1993;Fig. 3Ba). Interestingly, the T449E channel was as resistantto TEA block as the T449K mutant and had fast C-typeinactivation which was also unaffected by TEA (Fig. 3Bb).Thus, charged amino acids (either positively or negativelycharged) at position 449 prevent both TEA blockade and itscompetition with C-type inactivation.

B T449E

Control

\S, 70 mM K+

50 ms

b b

Control

1 nA

Control

0-3 nA

10 ms

Figure 3. Interaction of external K+ and TEA with the T449K and T449E mutant channelsA, superposition of currents generated during depolarizations to 0 mV applied to cells transfected with theT449K channel. A a, scaled current traces showing that the inactivation time constant (37 + 6 ms; n = 14)in the control solution was increased to 69 + 9 ms (n = 10) in 70 mm external K+. A b, lack of effect of70 mm external TEA on the inactivation rate. The inactivation time constant with TEA was 39 + 8 ms(n = 4). B, currents generated during depolarizations to 0 mV applied to cells transfected with the T449Echannel. Ba, scaled current traces showing that the inactivation time constant (26 + 7 ms; n = 13) in thecontrol solution was increased to 70 + 9 ms (n = 8) in 70 mm external K+. Bb, lack of effect of 70 mmexternal TEA on the inactivation rate. The inactivation time constant with TEA was 31 + 7 ms (n = 4).

J Phy8iol. 499.2 365

A. Molina, A. G. Castellano and J Lopez-Barneo

DISCUSSIONThis report describes how the interaction of TEA withamino acids in the pore region of Shaker B channels can beused to distinguish between the sites of TEA blockade andC-type inactivation. We have shown that TEA can bind toresidues located at the external entryway of the pore andblock the channels without altering inactivation. Thus, theconformational changes causing C-type inactivation must berather local and occur in a site deep within the outer mouthof the pore. These data give an insight into the mechanism ofC-type inactivation and contribute to an understanding ofthe structural basis for the interactions between ionoccupancy and gating in K+ channels.

In accord with previous work (MacKinnon & Yellen, 1990;Heginbotham & MacKinnon, 1992), our data indicate thattyrosine residues at position 449 of the Shaker B channelprovide for a high affinity TEA binding site. However, wealso show that the amino acid at location 447 is importantfor TEA binding, since the EC50 of the T449Y,D447Edouble mutant channel (-8 mM) is clearly higher than thatof the T449Y channel with a single mutation (-2 mM). Thefunctional role of aspartate in position 378 of Drkl channels,which is equivalent to position 447 of Shaker B K+ channels,has also been recently stressed (Kirsch et al. 1995).Therefore, it seems that, besides the aromatic side-chains oftyrosines at position 449, which are thought to form aphenolic cage with a high affinity for TEA (Heginbotham &MacKinnon, 1992), the carboxylates of the residue atposition 447 also contribute to the high affinity TEAbinding site (see Kirsch et al. 1995). On the other hand, theresistance of the T449E mutant to TEA block is noteworthy,given that TEA is a large cation that could be attracted bynegatively charged residues. It was suggested in previouswork that the effect on TEA block by the residue at position449 is not primarily by an electrostatic mechanism(MacKinnon & Yellen, 1990). However, the insensitivity toexternal TEA of channels bearing opposite electric charges(T449K and T449E) is particularly surprising and rules out

completely the existence of an electrostatic component inthe interaction of TEA with the outer region of the channelvestibule.

The various ways in which TEA interacts with the mutantchannels, summarized in Fig. 4, allow us to distinguishbetween the sites of TEA blockade and C-type inactivation.In the wild-type channel (T449T; Fig. 4A), TEA blocks thechannel with low affinity and in a voltage-dependentmanner, indicating that it moves deeply into the pore. Inthese conditions, TEA can interfere with the molecularmechanism of C-type inactivation (called for simplicity theC-inactivation gate). Baukrowitz & Yellen (1996) havereported that the site at which the last potassium ionoccupying the channel pore competes with C-typeinactivation senses about 50% of the electric field. Althoughthe affinity of the channel for TEA can be markedlymodulated by other residues, introduction of a tyrosine inposition 449 provides for a more superficial TEA bindingsite, essentially outside the transmembrane electric field(Fig.4B). In channels bearing this residue, TEA block ismore potent than in the wild-type channels but it does notinterfere with C-type inactivation. Finally, the introductionof a charged amino acid at position 449 (either lysine orglutamate; Fig. 4C) impedes the accessibility of TEA to thechannel vestibule and, consequently, to the C-inactivationgate. One could speculate that the residue at position 449,although not directly involved in inactivation, regulates theoccupancy of ions in deeper regions of the channel mouth.For example, a highly hydrophilic environment produced bylysine or glutamate at position 449 could interfere with thedehydration of cations necessary for their accessibility tothe narrow region of the conducting pore where theycompete with inactivation. This would explain why thesechannels have small conductances and fast inactivation ratesand are resistant to TEA block but highly sensitive toexternal K+.

It has been reported that the chemical modification ofsulfhydryl groups in amino acids at positions 448-450 is

BTEA blocks and competeswith C-type inactivation

CTEA blocks without (or withminor) effect on C-type inactivation

TEA does not block and is withouteffect on C-type inactivation

_E

Figure 4. Diagram of the outer mouth of potassium channels illustrating the separation betweenthe sites of TEA blockade and C-type inactivationA, wild-type channel (threonine at position 449). B, mutants bearing a tyrosine at position 449. C, mutantswith a charged residue (either lysine or glutamate) at position 449. See text for further explanation.

A

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Pore mutants, TEA block and C-type inactivation

highly state dependent, being favoured in the C-typeinactivated state (Yellen, Sodickson, Chen & Jurman, 1994;Liu et al. 1996; Schlief, Schonherr & Heinemann, 1996). Inthe mutant channel 448C it appears that disulphide cross-linking between subunits is favoured in the C-typeinactivated state (Liu et al. 1996). These last authors havesuggested that during C-type inactivation there is astructural rearrangement in the channel mouth resulting inexposure of the side-chains of residues 448-450 and a closerapposition of the four subunits. However, our findingsimpose strong limitations on this proposal. It seems unlikelythat the side-chain at position 449 lies buried in the restingor open states since substitution of this amino aciddrastically alters TEA binding and channel conductance(MacKinnon & Yellen, 1990; Pardo et al. 1992; Lopez-Barneo et at. 1993). Furthermore, if it is assumed thatC-type inactivation favours the exposure of the side-chain atposition 449, TEA should stabilize the C-type inactivatedstate in the mutant channels with tyrosine in this location.In contrast, we have observed that TEA can bind to thearomatic side-chains of tyrosine at position 449 withoutchanging C-type inactivation rates. These observationssuggest that the molecular rearrangement causing C-typeinactivation occurs near the entrance to the narrowestregion of the pore and that if there is a global motion of thefour subunits during inactivation it must be rather subtleand unable to distort the TEA binding site.

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AcknowledgementsWe thank Dr R. W. Aldrich for providing us with the Shaker BA6-46 cDNA. This work was supported by grants from theDGICYT (Spanish Ministry of Science and Education). A.M. is a

fellow of the FPI (Spanish Ministry of Science and Education).

Author's email addressJ. L6pez-Barneo: lbarneo@obelix.cica.es

Received 26 November 1996; accepted 3 January 1997.

inactivation in Shaker K+ channels: effects of alterations in thecarboxy-terminal region. Neuron 7, 547-556.

367J Phy8iol.499.2