©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The -Dendrotoxin Footprint on a Mammalian Potassium Channel (*)

(Received for publication, May 5, 1995; and in revised form, July 17, 1995)

Jan Tytgat (1) (2)(§) Tom Debont (1) Edward Carmeliet (2) Paul Daenens (1)

From the  (1)Laboratories of Toxicology, Van Evenstraat 4, and (2)Physiology, Campus Gasthuisberg, O and N, University of Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

alpha-Dendrotoxin, a 59-amino acid basic peptide from the venom of Dendroaspis angusticeps (green mamba snake), potently blocks some but not all voltage-dependent potassium channels. Here we have investigated the relative contribution of the individual alpha-subunits constituting functional Kv1.1 potassium channels to alpha-dendrotoxin binding. Three residues critical for alpha-dendrotoxin binding and located in the loop between domains S5 and S6 were mutated (A352P, E353S, and Y379H), and multimeric cDNAs were constructed encoding homo- and heterotetrameric channels composed of all possible ratios of wild-type and mutant alpha-subunits. Complete mutant channels were about 200-fold less sensitive for the alpha-dendrotoxin block than complete wild-type channels, which is attributable to a smaller association rate. Analysis of the bimolecular reaction between alpha-dendrotoxin and the different homo- and heteromeric channel constructs revealed that the association rate depends on the number of wild-type alpha-subunits in the functional channel. Furthermore, we observed a linear relationship between the number of wild-type alpha-subunits in functional channels and the free energy for alpha-dendrotoxin binding, providing evidence that all four alpha-subunits must interact with alpha-dendrotoxin to produce a high affinity binding site.


INTRODUCTION

Potassium channels of vertebrate neurons display a high degree of diversity that contributes to the complexity of function inherent to these cell types(1) . In the nervous system, potassium channels are crucial in controlling the resting membrane potential, repolarization of the action potential, and higher neuronal functions such as learning and memory(2) . The specific roles played by individual potassium channel types can be elucidated by taking advantage of differences in voltage-dependence, kinetics, and pharmacological specificity. Venom purification of certain snakes and scorpions has led to the isolation of peptide toxins, including potassium channel toxins, which are both selective and sensitive pharmacological tools to study the bountiful types of potassium channels (for a review, see (3) ).

First indications that the venom from the Eastern green mamba Dendroaspis angusticeps contains a peptide toxin which facilitates neuromuscular transmission came from studies of Barret and Harvey(4) . Separation of the venom into several components resulted in a particular interest of one polypeptide, named dendrotoxin, because it enhanced the quantal acetylcholine release at vertebrate neuromuscular junctions(5, 6) . The facilitation is most probably the result of high affinity binding to voltage-dependent potassium channels; in mammalian central neurones, dendrotoxin selectively blocks an inactivating voltage-dependent potassium current that corresponds to the 4-aminopyridine-sensitive ``A-type'' current(7) , and in mammalian peripheral neurons, dendrotoxin blocks slowly inactivating or noninactivating potassium currents(8) . Dendrotoxin constitutes about 2.5% of the total venom protein and is composed of 59 amino acids with a molecular mass of 7077 Da(5, 9) . In 1988, Benishin et al.(10) isolated four different polypeptides from the venom of the green mamba, designated as alpha-, beta-, -, and -dendrotoxin, and the amino acid composition of these four toxins indicated that alpha-dendrotoxin is identical to dendrotoxin (called DTX hereafter). (^1)Two similar polypeptides, called toxin I and K, have also been isolated from the venom of the black mamba Dendroaspis polylepis(11) , the former blocking rapidly inactivating, voltage-dependent potassium current in the frog node of Ranvier(12) . Although all of these toxins have a high degree of sequence homology with several protease inhibitors, such as the bovine pancreatic trypsin inhibitor, they do not share the pharmacological properties of protease inhibitors(13, 14, 15) . However, the tertiary structure of DTX is very similar to that of bovine pancreatic trypsin inhibitor, with two well defined regions of secondary structure, a double-stranded antiparallel beta-sheet and a short alpha-helical region at the amino terminus(16, 17) . DTX is a basic polypeptide with 8 arginine, 6 lysine, 2 aspartate, and 3 glutamate residues. At neutral pH it carries an overall positive charge, but the distribution of the charge is highly asymmetric. All members of the DTX family have 6 conserved cysteines, which might stabilize the structure through the formation of disulfide bonds(18) .

Because DTX blocks some but not all potassium conductances with high affinity, it has not only been useful in the pharmacological classification of potassium channels(19) , but also in the purification and isolation of potassium channel proteins(20) . The variation in toxin sensitivity exhibited by the expressed potassium channels cloned from Drosophila(21) and mammals (22) has provided the opportunity to correlate primary sequences of potassium channels with toxin sensitivity. Hurst et al.(23) have shown that DTX binding occludes the potassium channel pore by binding at or near the external mouth of the Kv1 channel at residues located in the loop between transmembrane domains S5 and S6. In this loop, residues alanine (Ala-352), glutamate (Glu-353), and tyrosine (Tyr-379), of the RBK1 (Kv1.1) channel were capable of influencing DTX blockage of the channel. The same authors also noted that through-space electrostatic forces play a role in DTX binding and concluded that the bound DTX must be stabilized by a number of residue interactions.

Given that the core of functional potassium channels is believed to be a tetramer comprising four alpha-subunits(24, 25, 26) , a mutation introduced in the cDNA coding for one alpha-subunit will result in a 4-fold effect at the level of the functional channel itself. When this mutation is critical for DTX binding and given that the toxin interacts with all 4 alpha-subunits, the macroscopic change in binding affinity will be determined by the presence of these four mutant subunits. So far, however, no data exist wherein the importance of each of the alpha-subunits for DTX binding is assessed in potassium channels. The present study reports experiments in which concatenated homo- and heteromultimeric RCK1 channel alpha-subunits, with all possible ratios of wild-type:mutant alpha-subunits (WT:MUT 4:0, 3:1, 2:2, 1:3, and 0:4), were expressed as functional potassium channels to study the contribution of each subunit to DTX binding and to establish whether all four alpha-subunits interact simultaneously with DTX.


MATERIALS AND METHODS

Construction and Expression of Wild-type and Mutant RCK1 Channels

Site-directed mutagenesis on the RCK1 potassium channel (27) was performed using the ``Transformer'' system (Clontech). Mutations were verified by dideoxy DNA sequencing (Sequenase Version 2.0, U. S. Biochemical Corp.). Construction of multimeric potassium channel cDNAs into a high expression vector, pGEM-HE, was as described previously(26) . For in vitro transcription, plasmids were first linearized with PstI (New England Biolabs) 3` to the 3`-nontranslated beta-globin sequence and then transcribed using T7 RNA polymerase and a cap analogue diguanosine triphosphate (Promega). Stage V-VI Xenopus laevis oocytes were isolated by partial ovariectomy under anesthesia (tricaine, 1 g/liter). Anesthetized animals were then kept on ice during dissection. The oocytes were defolliculated by treatment with 2 mg/ml collagenase (Boehringer Mannheim) in zero calcium ND-96 solution (see ``Solutions'' below). Between 2 and 24 h after defolliculation, oocytes were injected with 50 nl of 1-100 ng/µl mRNA. The oocytes were then incubated in ND-96 solution at 18 °C for 1-4 days. For each channel construct, at least two independent clones were transcribed, injected, and expressed.

Electrophysiology

Whole-cell currents from oocytes were recorded using the two-microelectrode voltage clamp technique. Voltage and current electrodes (0.4-2 megaohms) were filled with 3 M KCl or 1 M K(3)citrate plus 10 mM KCl. Current records were sampled at 4-ms intervals after low pass filtering at 0.1 kHz. Off-line analysis was performed on an IBM-compatible 80486 computer. Linear components of capacity and leak currents were not subtracted. Capacitative currents did not interfere with steady-state measurements of RCK1 currents, and leak current amplitudes were in the range of only approx1% of the amplitude of time- and voltage-dependent RCK1 currents. In noninjected or H(2)O-injected oocytes (n = 20), endogenous currents observed in the tested voltage range amounted to only approx1% of the amplitude of wild-type and mutant RCK1 currents. All experiments were performed at room temperature (19-23 °C) and a constant perfusion rate (2.5 ml/min). Steady-state blockade of the toxin was measured at the end of the test pulses (1 s) at different test potentials by stepping from a holding potential of -90 mV. Fitted K(d) values were obtained after calculating the percentage current left over by application of several DTX concentrations in different oocyte experiments (mean ± S.E., n).

Solutions

The ND-96 solution contained (in mM): 96 NaCl, 2 KCl, 1.8 CaCl(2), 1 MgCl(2), 5 Hepes, pH 7.5, supplemented with 50 mg/ml gentamycin sulfate (only for incubation). Lyophylized alpha-dendrotoxin (Alomone Labs) was dissolved in 1 ml (stock of 20 µM) and freshly diluted in the ND-96 solution just prior to (extracellular) bath application.


RESULTS

Hurst et al.(23) have shown that mutation of 3 residues in DTX-sensitive RBK1 channels, Ala-352, Glu-353, and Tyr-379, to match the equivalent positions in DTX-insensitive RGK5 channels, proline (Pro-374), serine (Ser-375), and histidine (His-401), causes a 150-fold decrease in DTX sensitivity. This provided a molecular explanation for the differences in DTX sensitivity observed among native potassium channels (Fig. 1A). Whether the molecular footprint of DTX binding is mainly determined by the interaction of the toxin with just one, or, in contrast, with all four alpha-subunits together, was not known. To investigate this we have made the same mutations A352P, E353S, and Y379H in an alpha-subunit of a RCK1 channel belonging to the Kv1.1 Shaker-type family. Next we constructed concatenated multimeric cDNAs composed of different combinations of WT and MUT alpha-subunits (Fig. 1B), allowing us to discriminate between two models of binding wherein the toxin either interacts with only one of the four alpha-subunits, or interacts simultaneously with all four alpha-subunits (see also below).


Figure 1: S5-S6 region of RBK1, RGK5, and RCK1. A, The S5-S6 region of the alpha-subunit of RBK1 beginning at residue Phe-346 and ending at Lys-386, and of RGK5 beginning at Phe-368 and ending at Lys-408. DTX sensitivity is crucially determined by residues Ala-352, Glu-353, and Tyr-379 in RBK1, and Pro-374, Ser-375, and His-401 in RGK5. Dashed horizontal lines represent identical residues. RBK1 is DTX-sensitive (gray ovals), whereas RGK5 is DTX-insensitive (black ovals). B, the S5-S6 region of the alpha-subunit of RCK1 beginning at Phe-346 and ending at Lys-386. MUT channels contain the A352P, E353S, and Y379H mutations. The Y379H mutation is supposed to reside in the H5 or pore region. A pictorial of concatenated homo- and heterotetrameric alpha-subunits is shown below, with DTX-sensitive (gray) and DTX-insensitive (black) alpha-subunits forming functional potassium channels.



Functional homo- and heteromeric RCK1 channels composed of all possible ratios of WT and MUT subunits revealed different pharmacological profiles for DTX block. Increasing concentrations of DTX were needed to achieve approximately the same extent of block, as WT subunits were substituted by MUT subunits (Fig. 2A). A constant concentration of 3 nM DTX blocked the currents through WT channels by about 80% and through channels composed of two WT and two MUT subunits by about 20%. The currents generated through complete MUT channels were practically unaffected at this toxin concentration (Fig. 2B).


Figure 2: Macroscopic currents of homo- and heteromeric channels. Current recordings obtained with the two-microelectrode voltage clamp technique. Oocytes were held at -90 mV and stepped to a test potential of 0 mV. A, currents through channels comprising 4:0, 3:1, 2:2, 1:3, and 0:4 WT:MUT alpha-subunits are shown both before and after application of increasing concentrations of DTX, leading to approximately the same extent of blockage. B, currents through complete WT, 2:2 WT:MUT, and complete MUT channels are shown both before and after application of 3 nM DTX. The current scale is 10 µA (Panel A) and 5 µA (Panel B).



The functional expression of the five different constructs, together with the fact that the mutations did not alter the kinetics, gating and potassium selectivity (V as a function of [K](o) not different between WT and mutant, data not shown), allowed us to test two models of DTX binding, one with and one without energy additivity. The model without energy additivity includes the following main features. (i) DTX binds on one of the alpha-subunits and occludes thereby the pore. (ii) There are four statistically distinguishable configurations available for a bound DTX molecule. (iii) When the channel has four identical subunits, such as the WT and MUT homotetramers, these four configurations will be energetically identical. (iv) When the channel has different sets of subunits, like the heterotetramers, then the interaction energy may be different for each configuration. (v) Channel inhibition can be described by a Langmuir adsorption isotherm. (vi) K(d) values for the heterotetrameric channels can be predicted in terms of the observed K(d) values of homotetrameric WT and MUT channels, according to

with K(i) values as microscopic binding constants. The model with energy additivity is characterized by the following main features. (i) DTX interacts simultaneously with all four alpha-subunits. (ii) A linear relationship exists between the free energy for DTX binding and the number of WT subunits. (iii) Channel inhibition can be described by a Langmuir adsorption isotherm. (iv) K(d) values for heterotetramers channels can again be predicted in terms of the observed K(d) values for homotetrameric WT and MUT channels, and is given by

The model without energy additivity predicts that, as long as at least one WT subunit is present, the channel will principally remain sensitive for DTX block. The model with energy additivity predicts equal spacing for the K(d) values on a logarithmic x axis for all the different channel constructs. Fitted K(d) values for block by extracellularly applied DTX of homomultimeric WT and MUT channels were 1.1 nM (n = 17 oocytes) and 199.3 nM (n = 25), respectively (Fig. 3A). Fitted K(d) values for heteromultimeric channels with 3:1, 2:2, and 1:3 WT:MUT subunits were 5.1 nM (n = 5), 17.6 nM (n = 26), and 84.4 nM (n = 7), respectively. These values correlate very well with the predictions of the model which includes energy additivity, as opposed to the model without energy additivity (Fig. 3B). A summary of the K(d) values, as fitted from the observed data and as predicted in models with or without energy additivity, is shown in Fig. 4. The semilogarithmic plot of the K(d) values as a function of the number of WT alpha-subunits reveals a linear relationship, as expected from the equal spacing of the K(d) values in Fig. 3B (right plot). The same linear relationship is also found on a linear plot between the free energy for DTX binding and the number of WT subunits (Fig. 4, right ordinate). Comparison between complete WT and complete MUT channels revealed that the mutations, A352P, E353S, and Y379H, result in channels that have a 200-fold decreased sensitivity for DTX, corresponding to a change in binding energy equivalent to more than 3 kcal/mol.


Figure 3: DTX sensitivity of multimeric constructs. A, dose-response curves for complete WT and MUT channels, with K(d) values of 1.1 and 199.3 nM, respectively (Hill coefficient of 1). Points are mean ± S.E. from 17 (WT) and 25 (MUT) oocytes. B, dose-response curves for complete WT and MUT channels as in Panel A (full lines), together with predicted dose-response curves (dashed lines) in a model without energy additivity (left) and with energy additivity (right). A superior prediction of the data is obtained in a model which includes energy additivity. Data points are mean ± S.E. from 5 (3:1 WT:MUT), 26 (2:2 WT:MUT), and 7 (1:3 WT:MUT) oocytes. For 2:2 WT:MUT channels, results obtained from tetrameric and dimeric cDNAs were pooled.




Figure 4: K(d) values and changes in binding energy. Semilogarithmic plot of K(d) values (left ordinate) and linear plot of mutant-induced changes in binding energy normalized to complete WT channels (DeltaDeltaG; right ordinate), as a function of the number of mutant alpha-subunits in the constructs. Open squares, predictions without energy additivity; open circles, predictions with energy additivity; filled circles, observations. The change in binding energy is given by DeltaG = -RT ln(1 M/K(d)). Lines were fitted by eye to the data.



Blockage induced by DTX showed no voltage-dependence, as the degree of block was not different in the range of test potentials from -30 to +40 mV (Fig. 5, A-C). The same extent of current inhibition was observed whether increasing concentrations of DTX were applied sequentially or cumulatively. Furthermore, DTX binding did not alter channel gating; the maximal conductance (g(max)) curve in control and 1 nM DTX conditions was characterized by a V value of -16.0 ± 3.1 mV (n = 10) and -16.5 ± 4.2 mV (n = 8), respectively, which is not significantly different (Fig. 5D). The steady-state inactivation was also not shifted by 1 nM DTX: -27.9 ± 1.4 mV (n = 5) and -28.9 ± 2.8 mV (n = 5) in control and DTX conditions, respectively. The block by DTX occurred rapidly and binding was reversible (Fig. 6A). The rapid onset and offset of the DTX action indicates an extracellular site of action.


Figure 5: Effect of DTX on voltage-dependence and gating. A, current recordings in control and 1 and 10 nM DTX conditions. Oocytes were held at -90 mV and stepped to test potentials varying between -80 and +40 mV. B, corresponding current-voltage (I-V) relationship, characterized by outward rectification typical for RCK1 channels. C, percentage current left over in 1 and 10 nM DTX conditions, as a function of V. No voltage-dependence was observed. D, corresponding maximal conductance-voltage (g(max)-V) relationship. No DTX-induced shift in the half maximal potential of activation (V) was observed (vertical dashed lines).




Figure 6: Bimolecular kinetics of DTX inhibition. A, an oocyte expressing WT channels was depolarized to 0 mV for 1 s from a holding potential of -90 mV every 5 s, both in the absence and presence of 3 nM DTX (control, open triangles; wash-in, open triangles plus black bar; wash-out, open circles). Values for and in this experiment were 14.7 and 72.3 s, respectively. B, bimolecular reaction scheme with C = channel, C:DTX = channel with bound DTX, k and k the apparent first-order association and first-order dissociation rate constants, respectively, alpha and beta the second- and first-order rate constants of association and dissociation, respectively, and the time constants for approach to equilibrium upon wash-in and wash-out, respectively.



Since DTX binding was reversible and did not alter channel gating, we next investigated whether DTX blockade followed a kinetic behavior of a simple bimolecular reaction. Current inhibition upon DTX application and recovery upon DTX removal followed a single exponential time course compatible with a bimolecular reaction scheme (Fig. 6B; for details, see legend). The effects of increasing DTX concentrations on the kinetics of block on a WT RCK1 channel are shown in Fig. 7A. As required by a bimolecular scheme, the apparent first-order association rate constant (k) increased linearly with toxin concentration, whereas the first-order dissociation rate constant (k) remained constant. Fig. 7B summarizes the second-order association rate constants (alpha) and the first-order dissociation rate constants (beta = k) of all the different channel constructs we have expressed (with ratios WT:MUT alpha-subunits ranging from 4:0 to 0:4). Gradually exchanging the four WT alpha-subunits by four MUT alpha-subunits, resulting in channels which become more and more insensitive for DTX block, can be attributed to a gradual decrease of the association rate constant. In contrast, the beta values remain constant for all channel constructs. Dividing the beta values by the alpha values for all the different channel constructs correlates well with the observed K(d) values (Fig. 4), which is in accordance with the equation K(d) = beta/alpha (Fig. 6B).


Figure 7: Association and dissociation rate constants. A, the apparent first-order rate constant of association (k) and first-order rate constant of dissociation (k) for DTX on a WT channel were calculated using the equations in 6B and plotted as a function of the DTX concentration. Each symbol represents an independent experiment. B, from similar measurements as those in Panel A, the second (alpha)- and first (beta)-order rate constants of association and dissociation were then determined for all the possible channel constructs (n geq 3) and plotted as a function of the number of WT alpha-subunits in the channel constructs.



Next, through-space electrostatic interactions between charged residues in RCK1 and DTX were examined by measuring the effectiveness of DTX to inhibit current in solutions of different ionic strength. For complete WT channels, the fitted K(d) was 1.1 nM (n = 17 oocytes) in normal ND-96 solution and 0.64 nM (n = 5) in an iso-osmotic solution containing only 48 mM NaCl (sucrose substitution). For complete MUT channels, the K(d) was, respectively, 199.3 nM (n = 25) and 43.6 nM (n = 6). This increase in sensitivity to DTX suggests that through-space electrostatic forces play a role in DTX binding, corroborating the results obtained by Hurst et al.(23) .


DISCUSSION

This study has identified that all four individual subunits of the RCK1 channel must interact simultaneously with a DTX molecule to produce a high affinity binding site. A similar mechanism of additive contributions from four tyrosine residues (Tyr-449) in the four subunits of Shaker-type channels has also been shown for the external TEA binding(26, 28) . The observation that each subunit in a symmetrical tetrameric channel is equally involved in the process of toxin interaction might be difficult to reconcile with the asymmetrical structure of a DTX peptide. However, the following points can be raised in accordance with our observations. If the overall dimensions of the toxin are much smaller than the outer channel vestibule, the binding process may be governed by a single toxin residue, annulling the importance of asymmetry of the peptide and interactions with other channel residues. The observed high affinity binding of DTX results then from the likelihood that a DTX molecule has ideally approached its binding site on the channel, as opposed to the frequent occurrence of collisions without binding (which have very large rate constants) between DTX and the potassium channel due to an inapt surface contact. For charybdotoxin (CTX), Garcia-Calvo et al.(29) have shown that the target size for the rat brain Kv1.3 CTX receptor is 253 kDa, which is about 4 times the size of a single pore-forming alpha-subunit of 58 kDa. The target size of 253 kDa implies that the toxin-binding site on Kv1.3 channels is lost as the channel tetramer is destroyed. This indicates a simultaneous interaction of CTX with all four subunits, similar to our results for DTX on Kv1.1 channels. In marked contrast with the foregoing, the CTX-binding site on the maxi-potassium channel activated by calcium seems associated with only a single alphabeta-subunit complex(29) . The finding of energy additivity of the four alpha-subunits for DTX binding does not allow us to conclude what type of molecular interaction is taking place. The observed free energy of DTX binding can be decomposed into a coulombic and a sterical term. As long as the three-dimensional structure of WT and MUT pore regions is not known, we can merely speculate which of the two terms is determinant. Additionally, interpretations of the free energy of toxin binding may be confounded by unknowable dehydration energies for residues that become dehydrated upon toxin binding, i.e. for toxin or channel residues that are buried in the region of intimate channel-toxin contact(30) .

This is the first report analyzing the contribution of each of the individual alpha-subunits to the DTX binding site in a functional potassium channel, based on the expression of multimeric cDNAs as opposed to co-injection experiments. Qualitatively, the use of homo- and heteromultimeric cDNA constructs is superior, since each type of tetramer encodes a homogeneous population of channels, at the same time controlling the ratio of WT:MUT monomers in the functional channel (see also (26) ). Based on co-injection experiments of WT and MUT Shaker potassium channels, MacKinnon (25) has demonstrated that the sensitivity for CTX, a 37-amino acid toxin from the scorpion Leiurus quinquestriatus quinquestriatus, depends on the ratio of WT:MUT subunits, and that the channels that include one or more MUT subunits have an intermediate CTX sensitivity, but that the WT CTX-sensitive phenotype is dominant. A recent report by Russel et al.(31) , however, suggests also that in heterotetramers from 2 Kv1 class potassium channels (Kv1.2 and Kv1.5), the CTX-insensitive monomer dominates the CTX pharmacology of the channel. From this we conclude that it is unlikely that the mechanism of blockage by DTX and CTX is the same (see also (32) ). The destabilized binding of DTX in MUT channels is expressed in a decreased association rate (alpha). Together with the finding that the four alpha-subunits interact simultaneously with a DTX molecule, this means that the mutations are expressed with equal energies in both the bound and transition states.

Although multimeric channel constructs have proven very useful in our previous studies (e.g. see (26) ), we performed an additional test for the validity of our multimeric constructs by screening the sensitivity of the hybrid channels for block by external TEA. The logarithm of the K(d) values of the homo- and heteromultimeric channels varied linearly with the number of WT subunits, indicating that also for the mutants in this study a TEA molecule is energetically stabilized in the pore of the channel by additive contributions from the four alpha-subunits(26, 28) . Furthermore, the currents generated through tetrameric 2:2 WT:MUT cDNA channel constructs were phenotypically indistinguishable from the currents through dimeric WT:MUT channel constructs, including their pharmacological profile.

Recombinant toxins and their associated large number of possible mutants constitute important new tools to delineate the site by which toxins recognize their multi-subunit targets. All residues of CTX have already been submitted to individual mutagenesis and it was found that the affinity changed dramatically when mutations were made at eight positions, among which three are positively charged residues, three hydrophobic, and two with hydrogen bonding capacity(33, 34) . Since CTX competes with TEA and DTX in various assays(3) , it was suggested that positively charged residues on DTX may also govern its binding. It was specifically tempting to anticipate that the lysine triplet (Lys-28, Lys-29, and Lys-30) on DTX may be associated with its specific binding, since residue Lys-27 on CTX appears to be located in the center of symmetry of the Shaker channel, playing a crucial role in blocking its pore(30) . However, using recombinant mutant DTX, Danse et al.(32) have provided evidence that the lysine triplet is unlikely to constitute a major element for the functional properties of DTX. Therefore, new DTX mutants are needed to clearly identify the residues by which DTX establishes intimate contact with RCK1 channels.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Research Associate of the Nationaal Fonds voor Wetenschappelijk Onderzoek (N.F.W.O., Belgium). To whom correspondence should be addressed. Tel.: 32-16-32-34-03; Fax: 32-16-32-34-05; jan.tytgat@med.kuleuven.ac.be.

(^1)
The abbreviations used are: DTX, dendrotoxin; WT, wild type; MUT, mutant; CTX, charybdotoxin; TEA, tetraethylammonium.


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