The aromatic binding site for tetraethylammonium ion on potassium channels

Neuron,

Vol. 8, 483-491, March,

1992, Copyright

0 1992 by Cell Press

The Aromatic Binding Site for Tetraethylammonium Ion on Potassium Channels Lise Heginbotham and Roderick Department of Neurobiology Harvard Medical School Boston, Massachusetts 02115

MacKinnon

Summary K+ channels are quite variable in their sensitivity to the pore-blocking agent tetraethylammonium ion (TEA) when it is applied to the extracellular side of the membrane. A Shaker K+ channel can be made highly sensitive by introducing a tyrosine (or phenylalanine) at residue 449 in each of the four subunits. A shift in the voltage dependence of blockade indicates that TEA senses a smaller fraction of the transmembrane electric field in the highly sensitive channels. There is a linear relationship between the free energy for TEA blockade and the number of subunits (zero, two, or four) containing tyrosine at 449, as if these four residues interact simultaneously with a TEA molecule to produce a high affinity binding site. The temperature dependence of blockade suggests that the interaction is not purely hydrophobic. These findings are consistent with a TEA-binding site formed by a bracelet of pore-lining aromatic residues. The center of the bracelet could bind a TEA molecule through a cation-x orbital interaction. Introduction Tetraethylammonium (TEA) ion and other quaternary ammonium (QA) ions inhibit K+ channels by binding within the ion conduction pore. These compounds have been used as probes to explore the pore entryways (Hille, 1967; Armstrong, 1971; French and Shoukimas, 1981), and for some K+ channels they have even been used to estimate the overall pore length (Miller, 1982; Villarroel et al., 1989). Most K+ channels are inhibited by QA ions applied to either the intracellular or extracellular surface. The internal and external inhibition sites are separate, and they appear to be located very near the pore opening on either sideof the membrane. Several experimental findings support these conclusions. First, QA blockade is only weakly voltage dependent, regardless of the side of application, as if the blocker enters only a short distance into the pore (Armstrong and Hille, 1972; Armstrong, 1971). Second, a recent mutagenesis study of the cloned Shaker K+ channel identified 2 specific residues that influence internal and external TEA blockade separately (Yellen et al., 1991). Finally, as we will discuss below, many of the functional properties of QA blockade from the two sides of the membrane are distinct (Armstrong and Hille, 1972; Armstrong, 1971; Hille, 1967). Nearly all K+channels are quite sensitive to internal blockade, and the specificity of the internal site for

different QA ions appears to be determined largely by the hydrophobicity of the blocker (Armstrong, 1971; French and Shoukimas, 1981). In general, the affinity increases with increasing hydrophobicity; blocker size per se appears to be less important. In contrast, with respect to external TEA sensitivity, most K+ channels fall into one of two classes: those that are sensitive (Ki < 1 mM) (Hille, 1967) and those that are relatively insensitive (Ki >20 mM) (Armstrong and Binstock, 1965). The higher affinity external site, unlike the internal site, is very selective for TEA over other QA compounds. Size, not overall hydrophobicity, appears to be the more important factor (Hille, 1967). Two recent studies have shown that one specific amino acid location in the pore-forming region of a K’ channel (position 449 of the Shaker K+ channel) is critical in determining the sensitivity to external TEA (MacKinnon and Yellen, 1990; Kavanaugh et al., 1991). Amino acids elsewhere in the pore-forming region can also influence the channel’s TEA sensitivity, but to a lesser extent (Yokoyama et al., 1989; Sttihmer et al., 1989; Frech et al., 1989; MacKinnon and Yellen, 1990); variation at position 449 alone can produce a IOO-fold change in the inhibition constant. Moreover, an aromatic residue at the 449 position appears to be a requirement for high affinity TEA blockade. The experiments described in this paper address the specific mechanism by which TEA interacts with the high affinity external binding site. We have found that 4 aromatic residues, 1 from each subunit, participate in the formation of the high affinity site. Moreover, we show that TEA interacts with all 4 of these aromatic residues at once, as if they “coordinate” the blocker in the pore. These findings identify a residue that lines the pore entryway, and they provide compellingevidencefortheexistenceof acation-rrorbital interaction between TEA and its aromatic binding site on the K+ channel. Results Tyrosine or Phenylalanine at Position 449 Produces High Affinity TEA Blockade Figure 1 shows the external TEA sensitivity of Shaker K+channels with different amino acids substituted at the 449 position (Figure 1). The TEA affinity depends critically upon the specific amino acid present. The graph shows that a high affinity site is produced only when phenylalanine or tyrosine is present at this position (K, = 0.35 m M and 0.65 mM, respectively). All other position 449 mutant channels studied (see legend to Figure I), including ones with the hydrophobic residues valine or isoleucine at this position, are at least 50 times less sensitive. Tyrosine and phenylalanine residues have in common a six-membered aromatic ring as the predominant structure of their side chain. These aromatic

Figure 1. The Effect of Different Acids at Position 449 on External finity

0.01

1

0.1 [TEA],

‘0 T/V’

rings evidently play a role in the interaction of a TEA molecule with the K+ channel. A K+ channel has four subunits, and in the experiments illustrated in Figure 1 the subunits are identical. Does a TEA molecule interact with a single one of these 449 residues at a time when it is blocking the channel, or does it interact with all four at once? Energy Additivity at the External TEA-Binding Site The experimental approach illustrated in Figure 2A addresses the following question. How is the TEA affinity affected when only two of the four subunits of a Shaker K+ channel have a tyrosine at position 449, while the remaining two subunits have a wild-type threonine residue? Hybrid channels were made by constructing a tandem dimer similar to one designed previously by lsacoff et al. (1990). Two tandem dimers are expected to participate in the formation of a tetramerit channel, as shown, with the.A and B halves across from each other. (See Experimental Procedures for the details of the dimer construction, along with a quantitative test of the model shown in Figure 2A.) Figure 2B shows the TEA inhibition results for channels with 0, 2, or 4 tyrosines (and therefore 4, 2, or 0 threonines, respectively) at the 449 positions. As we have already observed in Figure 1, a channel with 4 tyrosines (1 in each subunit) is inhibited with a K, of 0.65 mM. The same affinity is observed whether a channel arises from the coassembly of four independent monomers (each containing a tyrosine at 449) or from two dimers (with a tyrosine at the 449 position in both halves). Similarly, channels containing only threonine residues at position 449 are inhibited with the same affinity (K, = 22 m M ) regardless of whether they originate from four monomers or two dimers. When a tandem dimer has a tyrosine at position 449 in one half and a threonine in the other, the resulting channelswill have2position4!9tyrosinesand2threonines (Figure 2A). These hybrid channels display an intermediate sensitivity to external TEA, and the results are similar whether tyrosine is present in the A

I oc

Amino TEA Af-

TEA sensitivity of channels with different mutations at position 449 was measured as the fraction of original current (l/lo) rfmaining after applications of different concentrations of TEA. Measurements were madeusing two-electrodevoltageclampor outside-out macropatches. Oocytes were held between -60 and -80 mV, and currents were measured during steps to 0 mV. The amino acids substituted and studied at position 449 included phenylalanine!F), isoleucine (I), serine (S), valine (V), tryptophan (W), and tyrosine (Y), along with the wild-type threonine (T). Points and error bars represent the mean t S E M (when larger than points) of 3-7 measurements in separate oocytes.

or the B half of the dimer (Figure2). Thegraph in Figure 2B demonstrates a simple relationship between the K, for the hybrid channels and the K, values for each “parent” channel containing either 4 tyrosines or 4 threonines at the 449 positions. On a logarithmic plot, the curve for the hybrid channel falls exactly halfway between the curves for the two parent channels. Let us consider this result quantitatively. If we assume that there is a linear relationship between the free energy for TEA binding, AGO, and the number of subunits containing tyrosine at position 449, then we have

where the subscripts refer to channels with 0 (4T), 2 (2Y,2T), or 4 (4Y) position 449 tyrosines. Equation 1 leads directly to an expression relating measurable quantities, the TEA inhibition constants K: K2~,2:

= (K~YK~IT);‘~

(2)

The observed TEA inhibition constant for channels with 4 tyrosines, KAY, is 0.65 mM; that for channels with 0 tyrosines, K4~, is 22 mM. Equation 2 predicts an inhibition constant for the hybrid channel, K2Y,2T,of 3.8 mM. Indeed, the experimentally observed value is 3.7 m M (Figure 2B). The striking agreement between theexperiment and the prediction of Equation 2 offers insight into the way in which a bound TEA molecule may interact with the tyrosine residues at position 449. As we will discuss below (see Discussion), the finding of energy additivity argues that TEA must interact simultaneously with all 4 tyrosine residues. The Enthalpy of TEA Inhibition Is the interaction of TEA with its high affinity site predominantly hydrophobic? Hydrophobic binding reactions are frequently associated with a positive (unfavorable) enthalpy change that is “driven” by a large

The Aromatic 485

TEA-Binding

Site of K’ Channels

B

1.0

ITEA], TN Figure 2. Dependence

of TEA Affinity

on the Number

of Tyrosine

Residues

at Position

449

(A) A schematic diagram of channel formation with the dimeric constructs (see Experimental Procedures). (B) TEA sensitivity of channels with n = 0, 2, and 4 tyrosine residues at position 449. Using two-electrode voltage clamp, oocytes were held at a potential between -60 and -80 mV, and currents were elicited with a step to 0 mV. The fraction of current remaining after TEA application (I/lo) is shown as a function of the TEA concentration applied. Channels had either 4 threonine residues at position 449 (wild-type monomers [open diamonds], or dimers with T449 in both halves [closed diamonds]), 2 tyrosine and 2 threonine residues (dimers with the T449Y mutation in the first [closed circles], or second [closed triangles] half], or 4 tyrosine residues (T449Y mutant monomers [open squares], or dimers with the T449Y mutation in both halves [closed squares]]. Points and error bars represent the mean + SEM (when larger than points) of 3-7 measurements in separate oocytes.

positive change in entropy (Cantor and Schimmel, 1980). By measuring the temperature dependence of the equilibrium constant for TEA inhibition, the free energy can be decomposed into its enthalpic and entropic components. Figure 3 shows a van’t Hoff plot for external TEA inhibition of the T449Y K’ channel. The standard state enthalpy, determined from the slope of the graph, is about -25 KJlmol. This enthalpy value can fully account for the free energy of TEA binding (AGO = -18 KJlmol). In fact, the entropy component is actually unfavorable (about -0.06 KJ/mol * K). This finding does not tell us what kind of molecular interaction is taking place between TEA and its binding site on the channel, but it is certainly inconsistent with a pure hydrophobic effect.

79 !-

:e c

7: t

Specificity of the TEA-Binding Site Hille (1967) demonstrated that K+ channels in frog node of Ranvier are very sensitive to external TEA (Ki = 0.3 mM). He also showed that TEA is unique among the symmetric QA ions: larger and smaller derivatives are not effective inhibitors. The Shaker K+ channel with a tyrosine substituted at position 449 has a similar specificity. Figure 4 shows that tetramethylammonium ion (TMA) and tetrapropylammonium ion (TPA) inhibit the channel with low affinity. TMA is completely ineffective at the applied concentrations, and TPA inhibits with greater than IO-fold lower affinity

1 GOO,'temp Figure 3. TheTemperature consistent with a Purely

(1 /K)

Dependence of TEA Inhibition Hydrophobic Interaction

Is In-

TEA blockade of T449Y mutant channels was measured at several temperatures. The graph is a van’t Hoff representation of the data; each point represents a singlqexperiment. The slope is proportional to the standard state enthalpy change, AHO, for the TEA-channel interaction according to the thermodynamic relationship In(K) = -(AHO/RT) + (AWR], where K is the equilibrium association constant. A reasonable straight line yields an enthalpy value between -19 and -30 KJ/mol.

NWKJll 486

32.0

? ‘0 \

C6 _o \-

TPA

04

0 21

1

0

10

[QA].

Figure 4. Specificity

of the Binding

13C

n-iv'

Site

Blockade of the T449Y channel by different QA compounds is shown. TMA, TEA, and TPA were added to the bath solution. Some of these experiments were performed using two-electrode voltage clamp; others were done with outside-out macropatches. The membrane potential was held between -60 and -80 mV, and inhibition was measured during steps to 0 mV. Points and error bars represent the means f SEM of 3-7determL nations in separate oocytes.

comparedwithTEA.(Infact,TPAprobablyinhibitsthe channel at a different site. Many properties of TPA blockade are different from those of TEA, and TPA inhibition is the same whether or not tyrosine is present at the 449 position.) The specificity observed here suggests that the tyrosine residues at position 449 somehow form a size-selective binding site for an 8 8, TEA molecule, but not for the larger TPA or smaller TMA molecules.

Voltage Dependence of TEA Blockade A tyrosine residue at position 449 influences not only the affinity of external TEA for its receptor site, but also causes a marked shift in the voltage dependence of TEA blockade. Figure 5 shows a semi-logarithmic plot of the inhibition constant plotted as a function of membrane voltage for channels with 4, 2, or 0 (wildtype) tyrosine residues. When either 2 or 4 tyrosine residues are present, TEA senses only 4% of the transmembrane electric field. In contrast, TEA senses 19% of the field when blocking the wild-type channel with low affinity. The TEA molecule appears to reside at a more superficial location in the ion conduction pathway when it interacts with the tyrosine residues. In the absence of a high affinity site, a TEA molecule might enter more deeply into the pore (see Figure 7B).

Figure 5. The Presence of Tyrosine Residues at Position Alters the Voltage Dependence of TEA Blockade

449

Voltage dependence of TEA inhibition was studied in channels with 0 (circles), 2 (triangles), or 4 (squares) tyrosine residues ai position 449. Outside-out macropatches were made from oocytes expressing mutant channels. Patches were held at -80 mV, and currents were elicited by stepping the patch from -40 to f8Q mV in 10 mV increments. TEA inhibition was measured at each voltage in 4-6 separate patches. The voltage dependence observed in the wild type is consistent with TEA traversing 19% of the membrane potential difference, whi!echannelswith tyrosine at position 449 display nearly voltage-independent blockade.

Tyrosine Substitution at Position 449 Does Not Alter Other Channel Properties Figure 6 compares several properties of wild-type (threonine at position 449) and mutant channels with tyrosine substituted at position 449. The time course of activation and deactivation (Figures 6A and 6C) is similar and so is the voltage dependence of activation (Figure 6E). The channels also possess the same high selectivity for K+ over Na+ (Figures 6B and 6D), and the single-channel conductances are the same (Figure 6F). The T449Y mutation appears to be functionally silent: it influences only the channel’s sensitivity to external TEA. The pore diameter at the level of residue 449 is presumably about 8 A. A K ion will fit into a pocket of this size without having to shed its hydration shell. It is therefore not surprising to find that a threonine to tyrosine substitution here does not influence the conduction of K+ through the pore. Discussion Configuration of the TEA-Binding Site We have found that the free energy for TEA binding to its external binding site scales linearly with the

The Aromatic 487

TEA-Binding

Site of K’ Channels

B

A

Q c

2.0 1.5 1 .o 0.5 0.0 -0.5 -1 .o :

0.9 0.6 0.3 0.0 -0.3 -0.6 -0.9

0.3 0.0 -0.3 -0.6

0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 50

E

I 50

msec

msec

I 1 .o 0.8

0.0

$5 L&em@ -80 -60

I

I

I

-40

-20

0

I I -1 .o -10@75-50-25

mV Figure 6. Effect of the T449Y Mutation

on Channel

I 0

I

I

I

25

50

75

mV Gating

and Permeation

Properties

Inside-out macropatch recordings were made from oocytes injected with cRNA coding for either monomer wild-type channels ([A and B]; open circles), or dimer channels carrying the T449Y mutation in both halves ([C and D]; closed circles). (A and C) Patches were held at -100 mV and stepped for 50 ms to potentials from -40 to +50 mV in 10 mV increments. (B and D) Currents were elicited with a pulse from -100 to 0 mV for 50 ms and then stepped to tail potentials of -100 to 0 mV in 10 mV increments. (E) The voltage dependence of activation. (F) In patches containing only a few channels, the single-channel current was measured at several voltages for the two channel types. The two points at 0 pA are at the reversal potential for these channel types as determined within 5 mV by macroscopic tail currents (B and D). In all the experiments in this figure, internal (bath) solution contained 95 m M KCI, 1 m M MgCb, 5 m M EGTA, and 10 m M HEPES (pH 7.1); external (pipette) solution contained 20 m M KCI, 75 m M NaCI, 1.8 m M CaCI,, 1 m M MgCb, and 10 m M HEPES (pH 7.1).

number of subunits (zero, two, or four) containing a tyrosine at residue position 449. It is as if 4 tyrosines provide twice the favorable interaction energy provided by 2 tyrosines. The finding of energy additivity enables us to distinguish between two specific molecular pictures of the TEA-binding site. Let us begin by asking what effect the removal of 2 (out of 4) tyrosines should have if TEA interacts at its binding site with only 1 of the 4 position 449 residues at a time? In other words, imagine that there are four statistically distinguishable configurations available to a bound TEA molecule, and in each configuration TEA interacts with a position 449 residue from a single subunit. (These configurations may be separate bind-

ing sites on each subunit or they may be distinguishable positions of a TEA molecule within a single binding site. In either case, becauseTEA inhibits with a Hill coefficient of unity, we assume a I:1 stoichiometry for TEA binding to the channel.) If a channel has four identical subunits, then the four configurations will be energetically identical. If the subunits are not the same, for example at position 449, then the interaction energy may be different for each configuration. In general, these configurations may be in rapid equilibrium so that TEA can undergo transitions between its possible binding configurations without dissociating from the channel. For the case we are considering, the equilibrium reaction between TEA (T) and its four

bound written

configurations as

on the channel,

(T,C),,

can be

(3)

where K, (i = 1-4) are microscopic binding constants. Channel inhibition in this case is described by a Langmuir function of the TEA concentration, with an observed inhibition constant given by

Using Equation 4, we can express the expected inhibition constant for the hybrid channels, KZY,2~,in terms of the observed KdY and KdT: 2K4v &Y,ZT

=

___ KAY

+

KIT

6)

&T

We now have an equation relating the parent channels and the hybrid channel in the case in which TEA interacts with only a single position 449 residue at any moment in time. Substitution of the experimentally observed values for K4y (0.65 mM) and Kqi (22 mM) into Equation 5 predicts that K2Y,2Tshould be 1.3 mM. This value is inconsistent with the experimental results (K2Y,21 = 3.7 mM; Figure 2B). The observed inhibition constant, predicted by energy additivity, cannot be explained by a model in which TEA interacts separately with the position 449 residues and suggests that TEA must interact simultaneouslywith these residues. In a model in which TEA is able to shuttle from one configuration to the next, interacting with each of the position 449 residues separately, the interaction energies would not be averaged because TEA would spend most of the time in the more tightly bound configurations. But if TEA interacts simultaneously with the 4 tyrosine residues, and each diagonal pair contributes a fixed interaction energy, then the results of Figure 2 may be expected. It is generally assumed that TEA inhibits by occluding the pore, but for TEA added to the outside of a K+ channel, strong evidence for such a mechanism is lacking. However, the results here are most easily rationalized if TEA does block the pore. We find that the 4 position 449 residues interact simultaneously with an 8 A TEA molecule. These 4 residues must therefore lie near the central axis, where the four subunits come in contact. In other words, the side chains of the position 449 residues most likely line the ion conduction

pore. The weak voltage dependence of external TEA blockade would indicate that TEA binds at a superficial point along the axis of the pore, near the external entryway. Taken together, these findings suggest that amino acid 449 is located at the pore entryway on the extracellular side. The QA ion selectivity of the T449Y mutant channel argues that the pore diameter at the level of residue 449 is close to 8 A. Chemical Nature of the TEA-Binding Site What kind of interaction might be taking place between a TEA molecule and aromatic residues on the ion channel? A hint comes from a class of recently developed synthetic receptors that bind QA cations with high affinity(Doughertyand Stauffer, 1990; Shepodd et al., 1988). These receptors are essentially a bracelet of nonconjugated aromatic rings Iiningacentral cavity. QA cations sit in the cavity, where they interact favorably with the 71 electron orbitals of the aromatic rings. Noncharged analogs bind with much lower affinity (Stauffer and Dougherty, 1988), probably because an electronic interaction between the cationic ligand and the fixed quadrupole and inducible dipole of the aromatic groups is important. (Semiempirical calculations carried out by Sunner et al. [I9811 pointed to cation-fixed quadrupole and cationinduced dipole potentials as the predominant energy terms in the interaction between K’ and benzene.) If the tyrosines at position 449 line the entryway to the K+ channel pore and are able to lie flat against a somewhat cylindrical vestibule, then they may form a TEA-binding site that is very similar to the synthetic QA cation binding receptors. Figure 7A shows the hypothetical TEA-binding site that we have in mind, with a TEA cation in the cavity formed by four aromatic groups. This kind of TEA-binding site would beautifully account for many of the findings described here. Free energy additivity, a large negative enthalpy, and specificity based on size are all easily understood in terms of such an aromatic cage. Certainly, a cationaromatic interaction occurring in a protein is not a new concept. The recently determined crystal structure of acetylcholinesterase reveals an acetylcholinebinding site that is lined by aromatic residues (Sussman et al., 1992). Voltage Dependence It is interesting to compare the effect of mutations on TEA blockade in this study with the findings of Leonard and colleagues on the nicotinic acetylcholine receptor ion channel (Leonard et al., 1989). They found that mutations in the pore of the acetylcholine receptor altered the affinity of the pore blocker QX-222, but had little if any effect on the voltage dependence of blockade. They argued, based on their results, that the mutations must alter the energetics of the 4X-222 interaction, but not the location of the blocker in the pore. The situation here is very different. When the wild-type threonine residue at position 449 is replaced

The Aromatic 489

TEA-Binding

Site of K’ Channels

considering. The interaction between K’and benzene in the gas phase has been studied, and the conclusions are fascinating (Sunner et al., 1981). A K ion and a benzene molecule interact with nearly the same free energy as do K+ and a water molecule. In particular, the enthalpy of the benzene-K ion interaction is very favorable. Up to four benzenes were found to associate with a K ion. (A somewhat unfavorable entropy term was observed for the association of the third and fourth benzene molecule, presumably as a result of the limited degrees of freedom with which these can join the surrounding “solvent”cluster. In the pore of an ion channel, however, this entropy term should be less important, since the aromatic functional groups would be held in place to some extent by the protein.) Semi-empirical calculations for the benzene-K ion interaction in the gas phase led the authors to propose that a K ion sits at the center of a tetrahedral cage formed by four benzene molecules. One could imagine an aromatic binding site for inorganic ions in the pore of a K* channel similar to the TEA site depicted in Figure 7A. (These aromatic groups would probably not be oriented with the tetrahedral symmetry suggested for the gas phase interaction because of structural constraints). Within the region that is thought to form the pore, aromatic amino acid residues are found at three specific locations in all cloned K+ channels. One or more of these aromatic residues could potentially interact with conducting ions in the pore through a cation-x orbital interaction, much in the same way that tyrosine or phenylalanine at position 449 appears to interact with a blocking TEA molecule.

out

Figure 7. Cartoons

Depicting

the High Affinity

TEA-Binding

Site

(A) A TEA-binding site is formed by a cage of aromatic residues. (B) The high affinity site is formed by aromatic residues lining the entryway to the ion conduction pore. In channels displaying high sensitivity to TEA, blockade is less voltage dependent than in low affinity channels, as if theTEA-binding site is more superficially located in the high affinity channels.

by a tyrosine, not only is the affinity increased nearly So-fold, but the voltage dependence is decreased. The simplest interpretation is that the mutation alters the position of TEA in the pore: the high affinity binding site (on the T449Y channel) appears to trap the TEA molecule just at the entryway (figure 7B). In the absence of a high affinity site, TEA might enter more deeply into the pore and therefore be more voltage dependent. Implications for Ion Conduction The concept of cations interacting with aromatic I[: orbital electrons is intriguing. The residue at position 449 does not appear to be a critical ion conduction site in the K+ channel, but could cation-n orbital interactions be important for inorganic, permeant ions in a narrower region of the pore? We do not yet know theanswertothisquestion, but it isapossibilityworth

Experimental

Procedures

Molecular Biology The Shaker H4 K+ channel (Kamb et al., 1988) in a BlueScript vector (Stratagene) was modified by deleting amino acids 6-46 to remove fast inactivation (Hoshi et al., 1990). (However, the amino acid numbering corresponds to the unmodified Shaker H4.) Oligonucleotide mutagenesis was performed using thedutung- selection scheme of Kunkel (1985), and mutations were confirmed by sequencing the mutated region (Sanger et al., 1977). RNA was synthesized from Hindlll (NEB)-linearized DNA usingT7 RNA polymerase (Promega). In all cases but one (T449F), two independently produced clones were studied to minimize the chance that an observed phenotype was due to a stray mutation. Dimer constructs were made by engineering A (left half) and B (right half) protomers that could be mutated separately and then linked together. The A protomer has a short linking sequenceencodingan Ncol restriction endonucleasesiteaswell as 9 new amino acids (NNNNNNAMV) at the carboxyl terminus. Expressed alone, A protomer channels are indistinguishable from normal inactivation-removed Shaker K+ channels. The B protomer has a coding sequence identical to that of Shaker H4 (inactivation-removed), but there is a silent Ncol site at the starting edge of the amino terminus. The two protomers were joined in a single open reading frame by digesting each with Ncol and Hindlll, gel purifying, and ligating the Hindlll-Ncol fragment from A with the Ncol-Hindlll fragment from B. Electrophysiology Xenopus oocytes (Xenopus One, Ann Arbor, Ml) were prepared and injected with RNA as previously described (MacKinnon et

Neuron 490

al., 1988). Currents were recorded between days 1 and 5 after injectionfromwholeoocytesusingatwo-microelectrodevoltage clamp(OC-725, Warner Instruments) or from outside-out macropatches (Axopatch, Axon Instruments) from oocytes expressing the channels at a high density. The bath solution in all experiments, other than those illustrated in Figure 6, contained 96 m M NaCI, 2 m M KCI, 0.3 m M CaC&, 1 m M MgCI,, and 5 m M HEPES (pH 7.6). The pipette solution in macropatch experiments contained 95 m M KCI, 1 m M MgCI,, 5 m M EGTA, and 10 m M HEPES (pH 7.1). Currents were recorded before, during, and after the application of TEA to the extracellular side of the membrane. Testing Dimer Competence There are several imaginable ways that the dimer constructs could produce functional K+ channels. They might pair up in an ABAB fashion as we have suggested, but they might also contribute only one half (A or B) to a functional channel and leave the other half outside the channel complex. In this case, all combinations of channels, ranging from four A subunits to four 8 subunits, could occur. Finally, an AABB channel is also possible, since the linked carboxyl and amino termini connecting the two “cores”arequitelong. We havetestedthedimers inthefollowing ways. First, a dimer was produced with a point mutation in one half that makes that subunit resistant to a scorpion toxin (MacKinnon, 1991). The mutation renders a channel virtually insensitive to toxin only if all four subunits carry the mutation; even if only one subunit is wild type the channel is sensitive (with about a4foldloweraffinity).Thedimerchanneldisplaystheintermediate toxin sensitivity close to that expected based on the known interaction between the toxin and channels with fewer than four sensitive subunits. More importantly, the toxin sensitivity of the channel population is nearly uniform; most of the channels therefore are not produced by the random association of “dimer halves.” We have made a second test of the dimer constructions. By introducing a “lethal” mutation into one half of a dimer (for example, deletion of residues in the pore-forming region), we have found that functional (wild-type) channels do express, but to a level that is, at most, a few percent of “normal” dimers. It is possible that some “dimer half” association occurs, but only to a very small extent. The dimer construction results in a uniform population of channels (with respect to TEA sensitivity). Most likely the dimers behave as we expect, like half of a Nat or CaLA channel a subunit, although we cannot exclude the possibility that some AABB channels occur.

monium ion receptor in potassium vier. J. Gen. Physiol. 59, 388-400.

channels

of the node of Ran-

Cantor, C. R., and Schimmel, P. R. (1980). Biophysical Chemistry. Part 1: The Conformation of Biological Macromolecules (San Francisco: W. H. Freeman and Company), pp. 287-288. Dougherty, D. A., and Stauffer, D. A. (1990). Acetylcholine binding by a synthetic receptor: implications for biological recognition. Science 250, ?558-1560. Frech, C. C., VanDongen, A. M. J., Schuster, C., Brown, A. M., and Joho, R. H. (1989). A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning. Nature 340, 642-645. French, R. j., and Shoukimas, j. J. (1981). Blockage of squid axon potassium conductance by internal tetra-N-alkylammonium ions of various sizes. Biophys. J. 34, 271-291. Hille, B. (1967). The selective inhibition currents in nerve by tetraethylammonium 50, 1287-1302.

of delayed

potassium ion. j. Gen. Physiol.

Hoshi, T., Zagotta, W. N., and Aldrich, R. W. (1990). Biophysicai and molecular mechanisms of Shaker potassium channel inactivation. Science 250, 533-538. Isacoff, E. Y., Jan, Y. N., and Jan, L. Y. (1990). Evidence for the formation of heteromultimeric potassium channels in Xenopus oocytes. Nature 345, 530-534. Kamb, A., Tseng-C:ank, J., and Tanouye, M. A. (1988). Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron I, 421-430. Kavanaugh, M. J., Busch, tion between the pore of Biol. Chem.

M. P., Varnum, M. D., Osborne, P. B., Christie, A. E.,Adelman, J. P., and North, R. A. (1991). Interactetraethylammonium and amino acid residues in cloned voltage-dependent potassium channels. J. 266, 7583-7587.

Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA82,488492. Leonard, R. J., Labarca, C. C., Charnet, P., Davidson, N., and Lester, H. A. (1989). Evidence that the M2 membrane spanning region lines.the ion channel pore of the nicotinic receptor. Science 242, 1578-.1582. MacKinnon, R. (1991). Determination try of a voltage-activated potassium 235.

of the subunit stoichiomechannel. Nature 350, 232-

MacKinnon, R., and Yellen, G. (1990). Mutations blockade and ion permeation in voltage-activated Science 250, 276-279. We thank C. Miller and G. Yellen for their comments on the manuscript and D. Dougherty for helpful discussions. This work was supported by National Institutes of Health research grant CM43949. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

25, 1991, revised

December

2, 1991

affecting TEA KC channels.

MacKinnon, R., Reinhart, P. H., and White, M. M. (1988). Charybdotoxin block of Shaker K’channels suggests that different types of K’channelssharecommon structural features. Neuron ?,9971001. Miller, C. (1982). Bis-quaternary tural probes of the sarcoplasmic Physiol. 79, 869-891.

ammonium reticulum

Sanger, F., Nicklen, S., and Coulson, ing with chain-terminating inhibitors. 74, 5463-5467.

blockers as strucK’ channel. J. Gen.

A. R. (1977). DNA sequencProc. Natl. Acad. Sci. USA

Shepodd, T. J., Petti, M. A., and Dougherty, D.A. (1988). Molecular recognition in aqueous media: donor-acceptor and ion-dipole interactions produce tight binding for highly soluble guests. J. Am. Chem. Sot. 170, 1983-1985. Stauffer, D.A., and Dougherty, D.A. (1988). ion-dipole effect as a force for molecular recognition in organic media. Tetrahedron Lett. 29, 6039-6042.

Armstrong, C. M. (1971). Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J. Gen. Physiol. 58, 413-437. Armstrong, tion in squid ride. J. Cen. Armstrong,

C. M., and Binstock, L. (1965). Anomalous rectificagiantaxon injected with tetraethylammonium chloPhysiol. 48, 859-872. C. M., and Hille, 6. (1972). The inner quaternary am-

Stiihmer, W., Ruppersberg, J. P., Schroter, K. Ii., Sakmann, B., Stocker, M., Giese, K. P., Perschke, A., Baumann, A., and Pongs, 0. (1989). Molecular basis of functional diversityof voltage-gated potassium channels in mammalian brain. EMBO J. 8, 3235-3244. Sunner, J., Nishizawa, K., and Kebarle, P. (1981). Jon-solvent molecule interactions in the gas phase. The potassium ion and benzene. J. Phys. Chem. 85, 1814-1820.

The Aromatic 491

TEA-Binding

Site of K+ Channels

Sussman, J. L., Hare& M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991). Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholinebinding protein. Science 253, 872-879. Villarroel, A., Alvarez, O., Oberhauser, A., and Latorre, R. (1989). Probing a Ca2+-activated K’channel with quaternary ammonium ions. Pfliigers Arch. 473, 118-126. Yellen, G., Jurman, M., Abramson, T., and MacKinnon, R. (1991). Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel. Science 257, 939-941. Yokoyama, S., Imoto, K., Kawamura, T., Higashida, H., Iwabe, N., Miyata, T., and Numa, S. (1989). Potassium channels from NC10815 neuroblastoma-glioma hybrid cells: primary structure and functional expression from cDNAs. FEBS Lett. 259, 37-42.