Correspondence to: Kenton J. Swartz, Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg. 36, Rm 2C19, 36 Convent Dr., MSC 4066, Bethesda, MD 20892. Fax:301-435-5666 E-mail:swartzk{at}ninds.nih.gov.
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Abstract |
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Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 2025 Å away from the central pore axis.
Key Words: gating modifier toxin, scanning mutagenesis, voltage-dependent gating, proteinprotein interaction
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INTRODUCTION |
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Voltage-gated K+ channels are tetramers with each subunit containing six transmembrane segments, termed S1S6. As illustrated in Fig 1, we can think of these channels as constructed from two types of domains: a central pore domain formed by S5S6 and four surrounding voltage-sensing domains, each comprised of S1S4. The structure of the pore domain is likely to be similar to the crystal structure of the KcsA K+ channel, a bacterial channel that is homologous to S5S6 in voltage-gated K+ channels ( helices with the linker between them forming a short pore helix and the selectivity filter. A growing body of evidence suggests that the voltage-sensing domains are constructed from S1 through S4 (
helices (
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A large number of protein toxins isolated from venomous animals interact with voltage-gated ion channels and either physically block ion conduction or modify voltage-dependent gating. The pore-blocking toxins interact with the outer vestibule of the channel, as illustrated by the receptor for Agitoxin2 in Fig 1 B (
Hanatoxin is a protein toxin from spider venom that inhibits the drk1 voltage-gated K+ channel by binding to receptors on the extracellular face of the channel and modifying the energetics of gating (
In this paper, we studied the structural components and location of the Hanatoxin receptors on the drk1 K+ channel. First, we examined whether the three previously identified residues in the COOH-terminal end of S3 interact with the toxin by looking for patterns arising from multiple substitutions at each position. Second, we examined the possible contribution of additional residues in forming the Hanatoxin receptors by Ala-scanning a previously unexplored region between the NH2-terminal side of S1 and the COOH-terminal end of S3. Finally, we explored the possibility that Hanatoxin interacts with residues on the peripheral surface of the pore domain. Our results are consistent with an intimate interaction between Hanatoxin and channel residues in the COOH-terminal part of S3. In addition, our results suggest that the toxin does not interact with the extracellular surface of the pore domain, but rather exclusively interacts with the voltage-sensing domains of the channel.
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MATERIALS AND METHODS |
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Mutagenesis and Channel Expression
The cDNA encoding the wild-type drk1 K+ channel (
Oocytes from Xenopus laevis frogs were removed surgically and incubated with agitation for 11.5 h in a solution containing (mM): 82.5 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES, and 2 mg/ml collagenase (Worthington Biochemical Corp.), pH 7.6 with NaOH. Defolliculated oocytes were injected with cRNA and incubated at 17°C in a solution containing (mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, 50 µg/ml gentamicin (GIBCO BRL), pH 7.6 with NaOH for 15 d before recording.
Examination of the Toxin-Channel Interaction
Two-electrode voltage-clamp recording techniques were employed to study the toxin-channel interaction using an OC-725C oocyte clamp (Warner Instruments). Oocytes were studied in a 160-µl recording chamber that was perfused with a solution containing (mM): 50 RbCl, 50 NaCl, 1 MgCl2, 0.3 CaCl2, and 5 HEPES, pH 7.6 with NaOH. Data were filtered at 2 kHz (eight-pole Bessel) and digitized at 10 kHz. Microelectrode resistances were between 0.2 and 1.2 M when filled with 3 M KCl. All experiments were performed at room temperature (~22°C).
The equilibrium dissociation constant for Hanatoxin binding to closed or resting channels was determined using very negative holding voltages (-120 to -80 mV) where no steady state inactivation could be detected. The fraction of unbound channels was estimated using depolarizations that were too weak to open toxin bound channels and too short to perturb the equilibrium for toxin binding to resting channels, as previously described (
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This equation assumes four independent and equivalent binding sites on the drk1 K+ channel for Hanatoxin. For mutant channels with altered gating properties, all voltage protocols were adjusted appropriately so that the plateau phase in the I/I0voltage relation was well defined. The fraction of uninhibited current (I/I0) was initially measured for all mutant channels using between 100 and 250 nM Hanatoxin. The concentration dependence for toxin inhibition was further examined using two or three toxin concentrations for all mutants displaying >10-fold changes in toxin Kd in the initial screen.
Hanatoxin was purified from tarantula venom as previously described (
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RESULTS |
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Hanatoxin inhibits the drk1 voltage-gated K+ channel by shifting activation to more depolarized voltages (
Effects of Multiple Substitutions in the COOH-terminal Part of S3
Mutations to Ala at three positions in the COOH-terminal part of S3 in the drk1 K+ channel were previously reported to decrease Hanatoxin binding affinity by ~1025-fold (
The measurement of toxin binding affinity is illustrated in Fig 2 for the wild-type and two exemplary mutant channels (F274R and E277K). Voltage-activation relations were obtained in the absence and presence of various toxin concentrations using tail-current protocols (Fig 2A and Fig B). The fraction of uninhibited tail current was then measured for different strength depolarizations, as shown in Fig 2 C. This fraction of uninhibited current in the plateau phase at negative voltages (In/In0), which approximates the fraction of unbound channels, was used to calculate the Kd for toxin binding to the K+ channel (see MATERIALS AND METHODS) (
Fig 3 shows the results for multiple substitutions at F274, where mutation to Ala was previously reported to reduce Hanatoxin binding affinity by ~25-fold. The 14 substitutions made at F274 include 8 hydrophobic residues (Cys, Ala, Met, Pro, Tyr, Trp, Val, Ile), 2 basic residues (Lys, Arg), 2 acidic residues (Glu, Asp), Ser, and Gly. Fig 3 A shows the dependence of In/In0 on toxin concentration for the wild-type and two mutant channels, F274G and F274R. For the wild-type channel, the data were well described by a model assuming four equivalent and independent binding sites for Hanatoxin on the drk1 K+ channel with a Kd of 103 nM for toxin binding to each site (see MATERIALS AND METHODS). The two mutants, F274G and F274R, greatly reduced the toxin binding affinity with Kd values of 4.4 and 61.6 µM, respectively. The normalized Hanatoxin Kd values for all 14 substitutions at position 274 are summarized in Fig 3 B. One prominent trend in the data is that substitutions with hydrophobic amino acids cause the smallest perturbation in Hanatoxin binding energy. The range of G values [
G = -RT ln (Kdwt/Kdmut)] for hydrophobic residues (Ile, Val, Trp, Tyr, Pro, Met, Ala, Cys) is 1.21.8 kcal mol-1, while the range of
G values for nonhydrophobic residues (Gly, Asp, Ser, Glu, Arg, Lys) is 2.43.7 kcal mol-1. Although the hydrophobic residues caused the smallest perturbations, even these perturbations were quite significant, implying a very close and specific interaction with the toxin. These results are consistent with an intimate hydrophobic interaction between F274 and the toxin. It is also interesting that at position 274, substitutions with either Lys or Arg produce the largest perturbations, with
G values of 3.7 and 3.6 kcal mol-1, respectively. One possibility is that F274 is positioned close to a basic toxin residue in the channel-toxin complex (see DISCUSSION).
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We next examined the effects of multiple substitutions at E277. Fig 4 shows the concentration dependence of In/In0 for two mutant channels, E277Y and E277K. Compared with the wild-type channels (same data as for Fig 3), E277Y and E277K displayed greatly reduced binding affinities for Hanatoxin, with Kd values of 2.5 and 17.3 µM, respectively. The normalized Hanatoxin Kd values for all eight substitutions at position 277 are summarized in Fig 4 B. An interesting correlation between the magnitude of the effects and the substituted amino acids emerges. Substitutions with basic residues have the largest effects (G = 2.8 kcal mol-1), while those with neutral residues have more moderate effects (
G from 1.3 to 2.2 kcal mol-1). Mutation of E277 to Asp produced no discernible change in toxin binding affinity (
G = 0.04 kcal mol-1). The correlation between the charge of the side chain at 277 and toxin binding energy is to a first approximation consistent with a through-space electrostatic interaction between this position on the channel and a basic residue on the toxin. However, two observations argue for a more intimate interaction. First, substitutions with various neutral amino acids produce significantly different perturbations, with
G values ranging from 1.3 to 2.2 kcal mol-1. Second, the large values of
G for all mutations (1.32.8 kcal mol-1), with the exception of Asp, are more consistent with intimate or short-range interactions. We speculate that E277 interacts with a basic residue on Hanatoxin through formation of a salt bridge. In this case, the apparent tranquility observed upon substitution of Asp for Glu would imply some degree of conformational flexibility in either the toxin or the channel.
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The results of 13 substitutions at position 273 are shown in Fig 5. The mutation to Tyr produces the largest change in toxin binding affinity (G = 2.8 kcal mol-1). Substitutions with Thr, Arg, Asp, Ala, Gly, and Cys cause moderate changes (
G = 1.51.7 kcal mol-1) in toxin binding affinity, while substitutions with Val, Met, Trp, Phe, His, and Leu produce only minor changes (
G
0.5 kcal mol-1). These results are more complex than observed at either F274 or E277. The observation that most hydrophobic substitutions (Val, Met, Trp, Phe, and Leu) produced little change in toxin binding affinity with
G < 0.3 kcal mol-1 seems to hint at a hydrophobic interaction between position 273 and the toxin. However, the pattern is not simple because mutation to Tyr produces the largest perturbations (
G = 2.8 kcal mol-1) and very significant perturbations (1.6 and 1.7 kcal mol-1) were seen for mutation to Ala and Cys, two smaller hydrophobic residues. A partial explanation might be that I273 is involved in a hydrophobic interaction with the toxin and that there is a requirement for both a large and hydrophobic side-chain at position 273.
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Alanine-scanning Mutagenesis from the NH2-terminal part of S1 through the COOH-terminal Edge of S3
The above results are consistent with intimate interactions between three residues in the COOH-terminal end of S3 and Hanatoxin. While the surface area at the toxinchannel interface is unknown, the dimensions of Hanatoxin (~20 x 25 Å) argue that many channel residues, perhaps 10 or more, are located at the toxinchannel interface (
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Does Hanatoxin Interact with Residues on the Peripheral Surface of the Pore Domain?
From previous experiments, we know that Agitoxin2, a well-studied pore-blocking toxin, and Hanatoxin can simultaneously occupy the drk1 K+ channel (
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DISCUSSION |
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Previous studies support the idea that Hanatoxin interacts with four receptors on the voltage-gated K+ channel and inhibits the channel by modifying the energetics of gating (
In the first part of this paper, we examined the effects of multiple substitutions at three positions in the COOH-terminal end of S3, where mutations to Ala were previously found to alter toxin binding affinity. Our results for both I273 and F274 are consistent with hydrophobic interactions between these positions and hydrophobic residues on the toxin. The Glu residue at position 277 also seems to interact intimately with the toxin, perhaps by way of a salt bridge with a basic residue on the toxin. The solution structure of Hanatoxin was recently solved by NMR spectroscopy (-scorpion toxins and the sea anemone toxins. Hanatoxin and these Na+ channel toxins each have a face that contains a large hydrophobic patch surrounded by basic and acidic residues. This structural feature is illustrated in the surface rendering of Hanatoxin in Fig 8 A. Our results would be consistent with F274, and possibly I273, interacting with the hydrophobic cluster on the toxin. E277 might interact with one of the many basic residues (through formation of a salt bridge) that surround the hydrophobic patch. While our results with F274 are consistent with this residue participating in a hydrophobic interaction, they also show that the most dramatic perturbations result from mutations to either Lys or Arg. This might be an indication that in the toxinchannel complex, F274 is located close to a basic toxin residue, a quite feasible scenario for the face of Hanatoxin shown in Fig 8 A. It is interesting that the face of Hanatoxin proposed to interact with the channel resembles other well-studied proteinprotein interfaces. For example, the interface between growth hormone and its receptor shows a patch of hydrophobic residues, where tight hydrophobic contacts exist, surrounded by polar residues that participate in hydrogen bonds and salt bridges (
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The dimensions of Hanatoxin (~20 x 25 Å) strongly suggest that many channel residues, certainly more than three, are located at the interaction surface between toxin and channel. To search for additional components of the toxin receptors, we first systematically mutated the region spanning from the NH2 terminus of S1 to the COOH terminus of S3. All mutants in this region displayed binding affinities for Hanatoxin that are comparable to the wild-type channel (Fig 6). The other region that we examined for potential interactions with Hanatoxin was the extracellular surface of the pore domain. From the known structure of the KcsA K+ channel, we can infer that the extracellular surface of the pore domain in voltage-gated K+ channels has sufficient area to accommodate Agitoxin2, a pore-blocking toxin, and at least portions of four Hanatoxin molecules. When we mutated 18 residues (representing 72 positions in the homotetramer) on the peripheral surface of the pore domain, we found no significant alterations in Hanatoxin binding affinity (Fig 7). These results argue that Hanatoxin does not interact with the extracellular surface of the pore domain and therefore most likely interacts exclusively with the voltage-sensing domains. Since the extracellular surface of the KcsA K+ channel has dimensions of ~45 x 45 Å (
It is remarkable that mutations of only three residues in the entire voltage-sensing domain (S1S4) of the drk1 K+ channel have large effects on Hanatoxin binding affinity. One possibility is that Ala-scanning mutagenesis may have missed residues that contribute either weakly or moderately to the interaction with toxin because some substitutions to Ala represent rather conservative changes. Another very interesting possibility is that the toxin also interacts with the NH2-terminal part of S4, where a previous Ala-scan identified several positions where mutations have small effects (three- to fivefold) on Hanatoxin binding affinity (
Several recent studies examining the secondary structure of the first four transmembrane segments (S1S4) in voltage-gated K+ channels suggest that all four segments are membrane-spanning helices (
-helical character (
-helical structure for the COOH-terminal part of S3, there is evidence for
-helical structure in the nearby linker between S3 and S4 (
It is interesting that gating modifier toxins, perhaps as a general rule, interact with the equivalent region of voltage-gated K+, Na+, and Ca2+ channels. The present results are consistent with an intimate interaction between Hanatoxin and E277 in the drk1 K+ channel. Mutations at E1613 in the fourth repeat of the brain IIA Na+ channel alter the binding affinity of -scorpion toxin and sea anemone toxin, two types of toxins that slow inactivation (
1A voltage-gated Ca2+ channel disrupts the binding of
-Aga-IVA (Winterfield and Swartz, unpublished observations), a gating modifier toxin for the Ca2+ channel (
1A voltage-gated Ca2+ channel, also inhibits the drk1 K+ channel and the binding affinity of grammotoxin to the K+ channel is altered by mutations at I273, F274, or E277 (
1A Ca2+ channel. Another example is kurtoxin, an
-scorpion toxin that slows inactivation of the brain IIA Na+ channel and also inhibits T-type voltage-gated Ca2+ channels by shifting activation to more depolarized voltages (
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Acknowledgements |
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We thank Dave Hackos, Zhe Lu, and Rod MacKinnon for helpful discussions, Rosalind Chuang for making some of the mutants, and J. Nagle and D. Kauffman in the National Institute of Neurological Disorders and Stroke DNA Sequencing Facility.
Submitted: 6 January 2000
Revised: 5 April 2000
Accepted: 6 April 2000
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