Subunit Composition of Brain Voltage-gated Potassium Channels Determined by Hongotoxin-1, a Novel Peptide Derived from Centruroides limbatus Venom*

Alexandra KoschakDagger , Randal M. Bugianesi§, Jörg MitterdorferDagger , Gregory J. Kaczorowski§, Maria L. Garcia§, and Hans-Günther KnausDagger

From the Dagger  Institute for Biochemical Pharmacology, University of Innsbruck, Peter-Mayr Strasse 1, A-6020 Innsbruck, Austria and the § Department of Membrane Biochemistry & Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065

    ABSTRACT
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Procedures
Results
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Five novel peptidyl inhibitors of Shaker-type (Kv1) K+ channels have been purified to homogeneity from venom of the scorpion Centruroides limbatus. The complete primary amino acid sequence of the major component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide in Escherichia coli. HgTX1 inhibits 125I-margatoxin binding to rat brain membranes as well as depolarization-induced 86Rb+ flux through homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels stably transfected in HEK-293 cells, but it displays much lower affinity for Kv1.6 channels. A HgTX1 double mutant (HgTX1-A19Y/Y37F) was constructed to allow high specific activity iodination of the peptide. HgTX1-A19Y/Y37F and monoiodinated HgTX1-A19Y/Y37F are equally potent in inhibiting 125I-margatoxin binding to rat brain membranes as HgTX1 (IC50 values ~0.3 pM). 125I-HgTX1-A19Y/Y37F binds with subpicomolar affinities to membranes derived from HEK-293 cells expressing homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels and to rat brain membranes (Kd values 0.1-0.25 pM, respectively) but with lower affinity to Kv1.6 channels (Kd 9.6 pM), and it does not interact with either Kv1.4 or Kv1.5 channels. Several subpopulations of native Kv1 subunit oligomers that contribute to the rat brain HgTX1 receptor have been deduced by immunoprecipitation experiments using antibodies specific for Kv1 subunits. HgTX1 represents a novel and useful tool with which to investigate subclasses of voltage-gated K+ channels and Kv1 subunit assembly in different tissues.

    INTRODUCTION
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Introduction
Procedures
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References

Potassium channels comprise a large family of proteins that control electrical excitability as well as resting membrane potential in many different cell types. The discovery of peptidyl inhibitors in the venoms of different species such as scorpions, snakes, spiders, and sea anemone has played an instrumental role in the development of our current understanding of K+ channels. High affinity and selective peptides have been used to define the physiological role that K+ channels play in tissues of interest. As structural probes, they have guided the identification of the pore-forming region of these proteins. Radiolabeled derivatives of some of the peptides have been produced and used to identify receptor sites in native tissues, to purify these proteins to homogeneity from native tissues, and to determine their subunit composition (1-3). In addition, they have provided a means by which to develop the molecular pharmacology of K+ channels (4, 5).

In this study, we report the identification of five new peptides in the venom of the Central American scorpion Centruroides limbatus. The major peptidyl component, HgTX1,1 has been fully characterized and shares significant sequence homology with MgTX and noxiustoxin. A HgTX1 analog that can be radiolabeled without loss of biological activity has been produced by recombinant techniques and used to define the K+ channel subunit composition of the rat brain HgTX1 receptor. HgTX1, because of its potency and K+ channel pharmacology, represents a novel tool with which to study the distribution of K+ channel subtypes in different tissues.

    EXPERIMENTAL PROCEDURES
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Materials-- Venom from the scorpion C. limbatus was obtained from Miller International Venoms (Hollywood, FL). Escherichia coli DH5alpha was used for plasmid propagation, and strain BL21(DE3) was used for expression of the fusion protein. Plasmid PGEMEX-1 was from Promega. 125INa and 86RbCl were purchased from NEN Life Science Products. Restriction enzymes, Pfu DNA polymerase, T4 DNA ligase, nucleotide triphosphates, and reagents for polymerase chain reaction were obtained from Boehringer Mannheim. Glass fiber filters (GF/C) were from Whatman. Polyethyleneimine and bovine serum albumin (BSA) were purchased from Sigma. Iscove's modified Dulbecco's medium was from JHR Biosciences. HEK-293 cells stably transfected with homotetrameric Kv1 channels were obtained from Professor Olaf Pongs (Zentrum für Molekulare Neurobiologie, Hamburg, Germany). All other reagents were obtained from commercial sources and were of the highest purity grade commercially available.

Peptide Purification-- About 100 mg of crude C. limbatus venom was extracted and purified as described previously with minor modifications (7). Elution from the C18 reversed-phase column was achieved in the presence of a linear gradient of acetonitrile (0-35%, 78 min) at a flow rate of 1 ml/min. Purified peptides were reduced and subjected to Edman degradation as described (7).

Plasmid Construction, Synthesis, and Purification of Recombinant HgTX1-- The plasmid was created by altering pG9MgTX in the following manner: Ile2 right-arrow Val, Asn4 right-arrow Asp, Gln23 right-arrow Ile, and Ser24 right-arrow Arg. These mutations were introduced by the "gene SOEing" technique using mutagenic forward and reverse polymerase chain reaction primers (8). Desired mutations were verified by cDNA sequencing using the dideoxy chain termination method (9). The fusion protein was expressed in E. coli and purified as described by Koschak et al. (10). Composition of the purified material was verified by electrospray mass spectroscopy and Edman degradation. An identical procedure was used to generate and purify HgTX1-A19Y/Y37F.

Iodination of HgTX1-A19Y/Y37F-- A sample of HgTX1-A19Y/Y37F (38 µg; 9 nmol) dissolved in 100 µl of 100 mM sodium phosphate (pH 6.5) was incubated with 3-5 mCi of 125INa (30-50 µl) in the presence of IODO-BEAD® (1 bead; Pierce) for 10 min at room temperature. The bead was removed, and the reaction mixture was applied to a C18 reversed-phase column (Vydac; 0.45 × 25 cm) equilibrated with 0.05% trifluoroacetic acid. Elution was achieved in the presence of a linear gradient of acetonitrile in 0.05% trifluoroacetic acid (0-35% over 51 min). After lyophilization in the presence of 0.1% bovine serum albumin, the radioiodinated peptide was resuspended in 150 mM NaCl, 20 mM Tris-HCl (pH 7.4) and stored in small aliquots at -80 °C. The composition of the iodinated material was determined by automated Edman degradation and mass spectroscopy.

Membrane Preparation and Binding Studies-- Rat brain synaptic plasma membrane vesicles were prepared as described previously (5). Membrane vesicles from HEK-293 cells stably transfected with homotetrameric Kv1 channels were prepared as described (11). Binding experiments were carried out essentially as described previously (5). The incubation medium consisted of 20 mM Tris/HCl (pH 7.4), 0.1% bovine serum albumin (rat brain membranes) or 20 mM Tris/HCl (pH 7.4), 5 mM KCl, 0.1% bovine serum albumin (HEK-Kv1 membranes). Nonspecific binding was defined in the presence of 1 nM recombinant HgTX1 or MgTX, and incubation was carried out at 22-25 °C typically for 240 min. Experiments employing low receptor and/or radioligand concentrations (e.g. saturation studies) were allowed to reach equilibrium for >15 h. No detectable decay in receptor activity was observed after this incubation time.

Antibody Production and Immunoprecipitation Studies-- Polyclonal sera were raised against unique regions of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, and Kv1.6 channels (12). Rat brain membranes were incubated with 4-7 pM 125I-HgTX1-A19Y/Y37F at radioligand excess for >15 h at room temperature and solubilized as described previously (5, 13). Immunoprecipitation experiments were carried out in 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM KCl, 0.1% digitonin as described previously (5, 12, 13).

Efflux of 86Rb+ from HEK-293/Kv1 Cells-- HEK-293 cells stably transfected with homotetrameric Kv1 channels were plated into 96-well culture plates and maintained in Iscove's modified Dulbecco's medium with L-glutamine and HEPES). Cells were incubated overnight with 86Rb+ (3 µCi/ml). Depolarization-induced 86Rb+ efflux was measured as described previously (11).

Analysis of Data and Protein Determination-- Radioligand binding studies were analyzed as described (5). Protein concentration was determined according to Ref. 14 using bovine serum albumin as a standard.

    RESULTS
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References

Purification and Amino Acid Sequence of HgTXs-- Venom of the scorpion C. limbatus contains components that inhibit 125I-MgTX binding to voltage-gated K+ channels in rat brain synaptosomal plasma membrane vesicles. To isolate this inhibitory activity, ~100 mg of crude C. limbatus venom was fractionated on a Mono S HR10/10 cation exchange column. Two active fractions, eluting at ~0.18 M (peak 1) and ~0.20 M NaCl (peak 2), contained all this activity. They were loaded individually on a reversed-phase C18 column. The entire inhibitory activity of Mono S peak 1 was recovered in a single peptide (HgTX1), while peak 2 yielded several active fractions (HgTX2-5). The corresponding peptide sequences are shown in Fig. 1. They define HgTX1 as a 39-amino acid peptide with an overall amino acid sequence homology of 89% to MgTX (15) and 75% to noxiustoxin (16). Although only partial sequences of HgTX2 and HgTX3 were obtained, the data indicate that these two peptides also belong to families of previously identified K+ channel inhibitors. It is interesting that both HgTX2 and HgTX3 have one amino acid residue less between the third and the fourth Cys residue and therefore resemble more the family of ChTX/IbTX/Limbatustoxin/Leiurus toxin 2. 


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Fig. 1.   Primary amino acid sequences of HgTX1-5. The primary amino acid sequences of HgTX1-5 are presented. The residues mutated in HgTX1 to generate HgTX1-A19Y/Y37F are shaded.

HgTX1 inhibits binding of 125I-MgTX to rat brain membranes with an apparent Ki value of 0.31 pM but has no effect on 125I-IbTX-D19Y/Y36F binding to maxi-K channels (data not shown). This pharmacological profile of HgTX1 is identical to that of MgTX and suggests that HgTX1 is a novel peptide directed against voltage-gated K+ channels.

Expression and Oxidative Iodination of Recombinant HgTX1-- To verify that the amino acid sequence of HgTX1 corresponds to an active venom component, HgTX1 was expressed in E. coli and purified as described previously (10, 15). Recombinant HgTX1 inhibits 125I-MgTX binding to rat brain membranes with the same potency as native peptide (Ki = 0.47 pM). We failed to produce radioiodinated HgTX1 due to poor base-line separation of native and iodinated peptide. Therefore, we altered two residues in the synthetic HgTX1 gene; Ala was substituted for Tyr at position 19, and a Tyr to Phe conversion was introduced at position 37 (HgTX1-A19Y/Y37F). This approach has been successfully employed to radiolabel IbTX and AgTX1 (13, 17) and takes advantage of the fact that position 19 in these peptides can be modified without loss of biological activity because this residue does not form part of the peptide's interaction surface with K+ channels; only conservative substitutions at position 37 are tolerated. HgTX1-A19Y/Y37F was expressed and purified as described above for HgTX1. Binding of 125I-MgTX to rat brain membranes is inhibited by HgTX1 and HgTX1-A19Y/Y37F with identical Ki values (data not shown), suggesting that substitution of these two residues in HgTX1 does not cause any significant loss in toxin affinity. HgTX1-A19Y/Y37F was reacted with 125INa, and both the mono- and di-iodinated derivatives were well separated from native peptide (data not shown).

Interaction of HgTX1 with Homotetrameric Kv1 Channels-- Functional Effects of HgTX1 and HgTX1-A19Y/Y37F on homotetrameric Kv1 channels were investigated by monitoring depolarization-induced 86Rb+ efflux from HEK-293 cells that have been stably transfected with Kv1.1, Kv1.2, Kv1.3, or Kv1.6. Table I (top) summarizes these results. Both HgTX1 analogs inhibit with high affinities and similar potencies 86Rb+ flux through Kv1.1, Kv1.2, and Kv1.3 channels, whereas they are weaker inhibitors of Kv1.6. This pharmacological profile is identical to that seen with MgTX. It is worth noting that MgTX is indeed a high affinity inhibitor of Kv1.1, although 125I-MgTX does not bind to homotetrameric Kv1.1 channels transiently expressed in COS cells (12). These data suggest that iodination of MgTX at Tyr37 leads to a substantial modification of the peptide's pharmacological properties.

                              
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Table I
Pharmacological Characterization of HgTX1
Inhibition of 86Rb+ flux in HEK-293 cells, stably expressing different Kv1 channels by HgTX1, HgTX1-A19Y/Y37F, and MgTX is shown at the top. Inhibition of 125I-HgTX1-A19Y/Y37F binding to membranes derived from HEK-293 cells stably transfected with Kv1 channels by HgTX1, HgTX1-A19Y/Y37F, and MgTX is shown at the bottom.

Next, the binding properties of 125I-HgTX1-A19Y/Y37F to plasma membrane vesicles derived from HEK-293 cells expressing homotetrameric Kv1.1-Kv1.6 channels were examined. Incubation of these membranes with increasing concentrations of 125I-HgTX1-A19Y/Y37F leads to a concentration-dependent association of the peptide with Kv1.1, Kv1.2, Kv1.3, and Kv1.6 membranes (Fig. 2) but not with either Kv1.4 or Kv1.5 membranes. Binding to Kv1.1, Kv1.2, and Kv1.3 membranes occurs to a single class of receptor sites that display Kd values of 0.12, 0.17, and 0.21 pM, respectively, whereas binding to Kv1.6 membranes takes place with significantly lower affinity (Kd of 9.6 pM). The high affinity interaction of 125I-HgTX1-A19Y/Y37F with Kv1.1, Kv1.2, and Kv1.3 channels is due to very slow ligand dissociation at 22 °C (k-1 = 0.004-0.006 min-1; data not shown), whereas for Kv1.6 channels, the peptide displays much faster dissociation kinetics (k-1 0.14 min-1). HgTX1, HgTX1-A19Y/Y37F, and MgTX inhibit 125I-HgTX1-A19Y/Y37F interactions with these membranes with Ki values expected for this peptide's selectivity (Table I (bottom)). These data indicate that the pharmacological profile of 125I-HgTX1-A19Y/Y37F is identical to that of the unlabeled peptide (see Table I), making this ligand a more useful probe for investigating K+ channels than 125I-MgTX.


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Fig. 2.   Binding of 125I-HgTX1-A19Y/Y37F to homotetrameric Kv1 channels expressed in HEK-293 cells. Saturation binding analysis is shown. HEK-293 plasma membranes were incubated with 125I-HgTX1-A19Y/Y37F, and specific binding was assessed from the difference between total and nonspecific binding. Kv1.1 (Kd = 0.174 pM; Bmax = 0.188 pmol/mg protein), Kv1.2 (Kd = 0.118 pM; Bmax = 2.48 pmol/mg protein), Kv1.3 (Kd = 0.239 pM; Bmax = 3.91 pmol/mg protein), and Kv1.6 (Kd = 9.14 pM; Bmax = 0.49 pmol/mg protein) are shown. No specific radioligand binding was observed with Kv1.4 or Kv1.5 membranes. Insets, specific binding data are presented in the form of Scatchard representations.

Interaction of 125I-HgTX1-A19Y/Y37F with Rat Brain Synaptosomal Plasma Membrane Vesicles-- Since the specificity of HgTX1 for homotetrameric K+ channels was established, we next investigated whether the interaction of this ligand with rat brain membranes could be correlated with any given Kv1 channel. 125I-HgTX1-A19Y/Y37F binds to these membranes in a concentration-dependent manner (Fig. 3A) to a single class of binding sites that display a Kd of 0.15 pM and a Bmax of 2.3 pmol/mg of protein (n = 4).


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Fig. 3.   Binding of 125I-HgTX1-A19Y/Y37F to rat brain synaptosomal plasma membrane vesicles. A, saturation binding analysis. Rat brain membranes were incubated with 125I-HgTX1-A19Y/Y37F. For this experiment, a Bmax of 2.61 pmol/mg protein and a Kd value of 0.197 pM were determined. Inset, specific binding data presented in form of a Scatchard representation. B, association kinetics. Membrane vesicles were incubated with 0.18 pM 125I-HgTX1-A19Y/Y37F at 22 °C for the indicated periods of time. Nonspecific binding, determined in the presence of 1 nM HgTX1, has been subtracted from the experimental points. An association rate constant of 2.55×108 M-1 s-1 was determined. C, dissociation kinetics. After incubating membranes with 1.03 pM 125I-HgTX1-A19Y/Y37F for 120 min, toxin dissociation was initiated by the addition of 1 nM HgTX1 for different periods of time. A dissociation rate constant of 5.92×10-5 s-1 was determined. Inset, a semilogarithmic representation of the first order dissociation reaction.

The kinetics of 125I-HgTX1-A19Y/Y37F binding to rat brain membranes were determined. The data presented in Fig. 3B indicate that incubation of membranes with 0.18 pM 125I-HgTX1-A19Y/Y37F results in a time-dependent association of the peptide that approaches equilibrium after ~4 h. The mean association rate constant, k+1, was determined to be 8.2 × 108 M-1 s-1 (n = 3). This value is significantly higher than the diffusion control rate expected for a small peptide and suggests that long range electrostatic interactions occur between positive charges on the peptide and negative charges at the channel's outer vestibule. Dissociation of 125I-HgTX1-A19Y/Y37F from its receptor, initiated by the addition of excess HgTX1, displays single monoexponential kinetics with a k-1 of 1.8 × 10-4 s-1; (n = 3; Fig. 3C). The Kd calculated from these kinetic constants is 0.22 pM, a value very similar to the one determined under equilibrium binding conditions (0.15 pM).

To investigate the pharmacological properties of rat brain receptors labeled by 125I-HgTX1-A19Y/Y37F, structurally related (HgTX1, HgTX1-A19Y/Y37F, ChTX, MgTX, kaliotoxin, AgTX1, AgTX2, and IbTX) and unrelated (alpha -dendrotoxin) K+ channel peptide blockers were tested for their ability to modulate the binding reaction (data not shown). All peptides that had previously been characterized to interact with distinct subsets of voltage-gated K+ channels displaced radioligand binding with high affinity. The Ki values for these peptides are 0.2 pM (HgTX1), 0.22 pM (HgTX1-A19Y/Y37F), 0.31 pM (MgTX), 29.5 pM (ChTX), 2.1 pM (kaliotoxin), 0.21 pM (AgTX1), 0.94 pM (AgTX2), and 0.65 pM (alpha -dendrotoxin). Only IbTX, a selective blocker of maxi-K channels, had no inhibitory activity at concentrations up to 10 nM.

Subunit Composition of the Rat Brain HgTX1 Receptor-- To investigate in further detail the molecular components of the HgTX1 receptor, brain membranes were labeled with a saturating concentration of 125I-HgTX1-A19Y/Y37F and solubilized in the presence of digitonin. Solubilized receptors were subjected to immunoprecipitation experiments employing a complete panel of sequence-directed antibodies directed against individual Kv1 channels. The specificity of these antibodies has previously been determined to ensure that they exclusively recognize their corresponding antigens without displaying any cross-reactivity with other Kv1 subunits (5, 11, 12). All antibodies yielded saturable levels of precipitation (Fig. 4A), and, moreover, the presence of the corresponding competing peptide always decreased the level of precipitation by >90% (data not shown). Anti-Kv1.2 antibody precipitated all 125I-HgTX1-A19Y/Y37F receptors, and as a consequence, the combination of anti-Kv1.2 with any other anti-Kv1 antibody (e.g. anti-Kv1.1 or anti-Kv1.4) did not yield any increased level of immunoprecipitation. These results indicate that in virtually all brain HgTX1 receptors, Kv1.2 is an integral component. In the presence of anti-Kv1.1, 75 ± 5% of toxin receptors can be precipitated, whereas anti-Kv1.3 and anti-Kv1.6 only precipitated 17 ± 2% and 14 ± 4% of receptors, respectively. In the presence of saturating concentrations of both anti-Kv1.3 and anti-Kv1.6, additive precipitation levels (29 ± 2%) could be achieved (Fig. 4B), indicating that these two proteins are segregated into distinct HgTX1-sensitive channel complexes consisting of at least Kv1.2/Kv1.3 or Kv1.2/Kv1.6 subunits, respectively. Interestingly, anti-Kv1.4 consistently precipitated 45 ± 4% of receptor-bound 125I-HgTX1-A19Y/Y37F. Since homotetrameric Kv1.4 channels are not labeled by 125I-HgTX1-A19Y/Y37F (see above), and Kv1.2 subunits are always integral parts of the toxin receptor, Kv1.4 should be complexed with Kv1.2. The combination of anti-Kv1.1, anti-Kv1.3, anti-Kv1.4, and anti-Kv1.6 antibodies yielded the same precipitation level observed for anti-Kv1.2 alone, indicating that essentially no homotetrameric Kv1.2 channels are expressed in rat brain, and as a consequence, most Kv1.2 subunits should be assembled with other Kv1 subunits (see below). Only anti-Kv1.5 did not give any measurable level of toxin precipitation, suggesting that this Kv1 subunit does not contribute to the HgTX receptor in rat brain.


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Fig. 4.   Immunoprecipitation of digitonin-solubilized 125I-HgTX1-A19Y/Y37F receptors from rat brain by anti-Kv1 antibodies. A, digitonin-solubilized, 125I-HgTX1-A19Y/Y37F receptors were precipitated using increasing concentrations of anti-Kv1.1 (81.1%, open circle ), anti-Kv1.2 (100%, square ), anti-Kv1.3 (17.3%, black-square), anti-Kv1.4 (40.1%, black-diamond ), anti-Kv1.5 (<2%, *), or anti-Kv1.6 (10.1%, bullet ). One representative experiment is shown. The mean maximum level of precipitation of more than four independent experiments is given in B. B, the maximum level of precipitation by the indicated anti-Kv1 antibodies used alone (solid bars) or in combination (shaded bars) is shown. In all experiments, 10 µl of anti-Kv1.1, anti-Kv1.2, or anti-Kv1.3 or 20 µl of anti-Kv1.4, anti-Kv1.5, or anti-Kv1.6 was used for immunoprecipitation, and the overall amount of antibody was kept constant by the addition of preimmune serum.

To determine whether Kv1.3, Kv1.4, or Kv1.6 subunits could also be co-assembled with Kv1.1, we determined the additive effects of antibodies directed against these channels in the presence of anti-Kv1.1. The addition of anti-Kv1.3 yielded a significant additive effect as compared with anti-Kv1.1 alone (85 ± 3% sites are precipitated by combining these two antibodies), while anti-Kv1.4 together with anti-Kv1.1 resulted in 95 ± 4% precipitation. In contrast, anti-Kv1.6 did not increase the amount of receptor precipitated by anti-Kv1.1 alone (76 ± 6%). These data suggest that all 75% of 125I-HgTX1-A19Y/Y37F receptors in rat brain precipitated by anti-Kv1.1 alone should be composed by Kv1.1·Kv1.2 subunit assembly, while 15% of these Kv1.1·Kv1.2 oligomers are complexed with an additional Kv1.6 subunit. Kv1.3 subunits, however, are not likely to be co-assembled with Kv1.1 subunits due to the strict additivity of anti-Kv1.1 with anti-Kv1.3.

A very complex immunoprecipitation profile was observed when anti-Kv1.4 antibody was used together with anti-Kv1.1. This antiserum precipitated 45% of toxin receptors when used alone, but it significantly increased the level of anti-Kv1.1 precipitation by an additional 20 ± 4% to yield 95% precipitation (as compared with the effects of anti-Kv1.1 alone). Thus, this additional 20% of toxin receptors precipitated by anti-Kv1.4 antiserum should be devoid of Kv1.1, since these antisera are partly additive, and may represent Kv1.2·Kv1.4 heteromultimers (eventually in combination with Kv1.3; see below). The remaining 25% of Kv1.4-containing sites, which do not show any additivity with anti-Kv1.1, are most likely assembled in a Kv1.1·Kv1.2·Kv1.4·Kv1.x oligomer, with this putative Kv1.x subunit representing either a second copy of an established subunit or a yet unidentified additional Kv1 subunit. The combination of anti-Kv1.3 and Kv1.4 resulted only in a very slight increase of precipitation when compared with anti-Kv1.4 alone, indicating that these two subunits could partly be assembled in a complex together with Kv1.2 (these data not shown).

Taken together, all of these data suggest that about 30% of rat brain 125I-HgTX1-A19Y/Y37F receptors are formed by exclusive assembly of Kv1.1 with Kv1.2, while 30% of toxin receptors appear to possess an additional Kv1.4 subunit occurring together with Kv1.1 and Kv1.2. Only a minority of sites (15%) appear to be composed of Kv1.1·Kv1.2·Kv1.6 subunits, and some receptors are likely to be formed by Kv1.2·Kv1.3·Kv1.4 assembly. However, the absolute number of subunits within any given Kv1 channel complex cannot be determined from these immunoprecipitation experiments, since the presence of a single subunit would yield the same level of precipitation as multiple subunit copies.

    DISCUSSION
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Procedures
Results
Discussion
References

HgTX1 Is a High Affinity Ligand for Voltage-gated K+ Channels-- In this study, we report the purification and characterization of a novel K+ channel-blocking peptide, HgTX1, from the venom of the Central American scorpion C. limbatus. Recombinant HgTX1 inhibits with high affinity 125I-MgTX binding to rat brain membranes but not 125I-IbTX-D19Y/Y36F binding to maxi-K channels, suggesting that HgTX1 is a selective blocker of voltage-dependent K+ channels. The specificity of HgTX1 for members of the Kv1 family indicates that this peptide inhibits with equivalent high potency Kv1.1, Kv1.2, and Kv1.3 channels, but it displays lower affinity for Kv1.6. This pharmacological profile is identical to that of MgTX (Table I). However, it was shown in previous studies that 125I-MgTX does not bind to homotetrameric Kv1.1 channels transiently expressed in COS cells (12). These data suggest that iodination of MgTX at Tyr37 alters the peptide's pharmacological properties. Since Tyr37 is presumed to form part of the peptide's interaction surface with the channel, covalent modification of that residue could lead to alteration in the conformation of the peptide and, as a consequence, to a different pharmacological profile. The bulky hydrophobic iodine at Tyr37 might also prevent an interaction with residues located in the pore of the channel. To circumvent this problem, HgTX1-A19Y/Y37F was prepared and shown to display the same functional characteristics as HgTX1. In contrast to 125I-MgTX, which only binds with high affinity to Kv1.2 and Kv1.3 channels, binding of 125I-HgTX1-A19Y/Y37F occurs with subpicomolar dissociation constants to Kv1.1, Kv1.2, or Kv1.3 channels. However, interaction with Kv1.6 channels is of lower affinity, and the peptide does not bind to either Kv.1.4 or Kv1.5 channels. Thus, iodination of HgTX1-A19Y/Y37F does not alter the pharmacological properties of the peptide and yields a very high affinity ligand selective for a subset of voltage-gated K+ channels.

Subunit Composition of Rat Brain Voltage-gated K+ Channels Labeled by 125I-HgTX1-A19Y/Y37F-- In rat brain membranes, 125I-HgTX1-A19Y/Y37F binds to a single class of binding sites with a dissociation constant identical to that determined for homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels. Thus, the brain receptor is likely to be associated with either one or all of these Kv1 channels. To investigate in further detail the molecular components of the rat brain HgTX1 receptor, we employed antibodies specific for individual Kv1 subunits in immunoprecipitation studies of detergent-solubilized membranes that had been labeled with 125I-HgTX1-A19Y/Y37F. All HgTX1 receptors were found to contain at least one Kv1.2 subunit. A number of putative complexes containing two or three distinct subunits (e.g. Kv1.1·Kv1.2, Kv1.2·Kv1.4, Kv1.1·Kv1.2·Kv1.4, Kv1.1·Kv1.2·Kv1.6, and Kv1.2·Kv1.3·Kv1.4) were unequivocally identified. Neither Kv1.1, Kv1.2, nor Kv1.3 appears to exist as a homooligomer. However, we cannot exclude the possibility that either yet unidentified additional subunits or several copies of one subunit are contained in any of the identified K+ channel complexes. Our findings are in agreement with previously published immunohistochemical distribution studies where Kv1.1·Kv1.2 or Kv1.2·Kv1.4 subunits have been found to be co-localized in specific regions of the rat brain (12, 18-21).

Heterooligomeric association of Kv1 subunits has recently been reported in bovine cerebral cortex (22) by immunoprecipitation experiments and subsequent identification of the channel subunits by Western blotting. However, a quantitative determination of the relative abundance of each individual oligomer was not possible using this approach. Moreover, denaturation of channel oligomers followed by subunit dissociation could have hampered the precise detection of naturally occurring subunit combinations. It is worth mentioning that our precipitation experiments also have the limitation that only complexes to which radiolabeled peptide remains bound at the end of the immunoprecipitation assay can be detected. For instance, homooligomeric Kv1.4 and Kv1.5, channels to which HgTX1 does not bind, would not be detected by this approach. The same is true for putative heterooligomers of Kv1.5 with other HgTX1-sensitive subunits, since it has been shown that Kv1.5 would confer a dominant phenotype to these complexes (23). Even homotetrameric Kv1.6 channels, to which HgTX1 binds with much lower affinity, would be difficult to identify because of significant ligand dissociation during the time course of the study. The development of new ligands that display high affinity interaction with the different Kv1 subunits could therefore be of great interest for defining the total subunit composition of these channels. Regardless of the limitations discussed above, HgTX1 represents a very useful tool for identifying subsets of voltage-gated K+ channels in different tissues.

    ACKNOWLEDGEMENTS

We thank Maria Trieb, Emanuel Emberger, and William Schmalhofer for technical contributions and Tracey Klatt for mass spectroscopy studies. We gratefully acknowledge Dr. Robert Koch for experimental support in the early stage of the project and Drs. Jörg Striessnig, Robert Slaughter, and Hartmut Glossmann for continuous discussion.

    FOOTNOTES

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

A preliminary report of this work has been presented in abstract form (6).

Supported by Austrian Research Foundation Grants S6611-MED and P-11187-MED, Austrian National Bank Foundation Grant 6239, and the European Union BIOMED 2 program Grant BMH4-CT96-2118. To whom correspondence should be addressed. Tel.: 43-512-507-3156; Fax: 43-512-507-2858; E-mail: hans.g.knaus{at}uibk.ac.at.

1 The abbreviations used are: HgTX, hongotoxin; ChTX, charybdotoxin; 125I-ChTX, monoiodotyrosine charybdotoxin; MgTX, margatoxin; 125I-MgTX, monoiodotyrosine margatoxin; IbTX, iberiotoxin; Kv1 channel, voltage-gated K+ channel, Shaker-type; BSA, bovine serum albumin; AgTX, agitoxin.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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