[32] Scorpion toxins as tools for studying potassium channels

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TOXINS AND OTHER MEMBRANE ACTIVE COMPOUNDS

[32]

tration. Iodinated peptides may also stick to dust particles introduced into solution, for example, by pipette tips. This can lead to scatter of signal in receptor binding assays. To avoid this, stock solutions of radiolabeled peptide are centrifuged (e.g., in an Eppendorf microfuge) to pellet such particles prior to solution use. 65 p. Hess, Annu. Rev. Neurosci. 13, 337 (1990). 66 R. W. Tsien, P. T. Ellinor, and W. A. Horne, Trends Pharmacol. Sci. 12, 349 (1991). 67 T. P. Snutch, Curr. Biol. 2, 247 (1992). 68 S. Sato, H. Nakamura, Y. Ohizumi, J. Kobayashi, and Y. Hirata, F E B S Lett. 155, 277 (1983). 69 E. Moczydlowski, B. M. Olivera, W. R. Gray, and G. R. Strichartz, Proc. Natl. Acad. Sci. U.S.A. 83, 5321 (1986). 7o M. M. Stephan, J. F. Potts, and W. S. Agnew, J. Membr. Biol. 137, 1 (1994). 71 M. Fainzilber, D. Gordon, A. Hasson, M. E. Spira, and E. Zlotkin, Eur. J. Biochem. 202, 589 (1991). 72 y . Hidaka, K. Sato, H. Nakamura, J. Kobayashi, Y. Ohizumi, and Y. Shimonishi, F E B S Lett. 1, 29 (1990).

[32] S c o r p i o n T o x i n s a s T o o l s f o r S t u d y i n g Potassium Channels By MARIA L. GARCIA, MARKUS HANNER, HANs-GIJNTHER KNAUS,

R O B E R T SLAUGHTER,

and G R E G O R Y J. KACZOROWSKI

Introduction Ion channels play a fundamental role in control of cell excitability. Thus, their activity is largely involved in modulation of contractility of muscle cells, and in release of hormones and neurotransmitters from endocrine and neuronal cells. Out of all the families of ion channels, K + channels represent the largest and most diverse group of proteins. Gating of these proteins occurs through conformational changes that are controlled by voltage and/or ligand binding. Therefore, K + channels can be broadly divided into two groups: voltage-dependent and ligand-activated channels. A number of techniques have become available during the last few years for studying ion channel structure and function. Electrophysiology affords determination of biophysical parameters that are inherent to each individual ion channel. With the use of molecular biology, a large amount of information regarding the structure and existence of subfamilies of K + channels has become available due to molecular cloning of cDNAs encoding these

METHODS IN ENZYMOLOGY,VOL. 294

Copyright © 1999by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/99 $30.00

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proteins. 1 However, two major questions are still the subject of further investigation. These concern the molecular composition of given channels as expressed in vivo, as well as the physiologic role that channels play in cell function. To explore these questions, high-affinity, selective modulators for a given channel must be found. Progress in this area has occurred due to discovery of high-affinity peptidyl inhibitors of K + channels in venom of different organisms, such as scorpions, snakes, bees, spiders, and sea anemone. 2 These peptidyl inhibitors have been useful in development of the pharmacology of K + channels, and have also been employed in binding reactions as a marker during the purification of channels from native tissues. This has allowed determination of a channel's subunit composition, as well as identification of specific auxiliary subunits of K + channels. These auxiliary proteins are very important for channel function because they cause profound effects on both the biophysical and pharmacologic properties of the pore-forming subunit. In addition, due to their well-understood mechanism of action, peptidyl inhibitors of K + channels have allowed the identification and molecular characterization of the pore-forming region of these proteins, and determination of subunit stoichiometry. Scorpion venoms constitute a rich source of peptidyl inhibitors of K + channels. 2 These peptides typically consist of 37-39 amino acids and display significant sequence homology among themselves. They usually contain six Cys residues that form three disulfide bridges, thus providing the peptide with a very rigid structure. Production of significant quantities of these peptides can be accomplished by either solid-phases synthesis or recombinant techniques. This has allowed determination of the three-dimensional solution structure of some of these peptides by nuclear magnetic resonance (NMR) techniques. The overall structures so far obtained are very conserved and possess an o~-helical region that is linked by disulfide bonds to a three-strand antiparallel /3 sheet. Knowledge of the three-dimensional structure of these peptides, combined with site-directed mutagenesis, has allowed identification of those residues critical for binding to the channel pore, and have provided a picture of the interaction surface with K + channels. Importantly, all residues that are crucial for interaction with the channel are located on one face of the/3 sheet, while the o~-helical region does not make contact with the channel directly. Complementary mutagenesis targeting residues in the channel pore has then allowed derivation of models describing shape and dimensions of the receptor in the vestibule of the channel. 1 A. Wei, T. Jegla, and L. Salkoff, Neuropharmacology 35, 805 (1996). 2 M. L. Garcia, M. Hanner, H.-G. Knaus, R. Koch, W. Schma|hofer, R. S. Slaughter, and G. J. Kaczorowski, Adv. Pharmacol. 39, 425 (1997).

626

[32]

TOXINS AND OTHER MEMBRANE ACTIVE COMPOUNDS

NVS

TTSK

RLHNTSRG

K

NK

Lq2

ZF ZF ZF

DVD QES

SV SK TASN

D L F G V D RG RLHNTNRG

K K

GK NK

YS YQ YS

LbTX

VF

DVS

SV SK

AAVGTDRG

K

GK

Y...

NxTX

T I T I

NVK NVK

TSPKQ~SKP TSPKQ~LPP

K E L YGSSAGA KAQFGQSAGA

K K

IT VF

NVK NAK

T S P Q Q ~ L RP RGSP E iLP K

KDRFGQHAGGK KEAIGKAAG

NG NG NG

K

NG

GV P

TGSP TGSP

LKP ~IKP

ChTX

IbTX

Mg'rX C.I,I. I TyK~

M

rPl~

Ki !M

I-pl~ l-Pl~

K~ i

GMRFG GMRFG

K K

GMRFG GMRFG

AgTX2

GV P

AgTX3

GV P

NVP

TGSP

IIKP

KTX

GVE

NVK PVS

SGSP KHSG

LKP LKP

KDA KDA

V R

I-PI~ TPI~

GMRFG

iKDA IKDA

KTX2

i,:M

KDA

NVK NVS

AgTX1

YNb YPI" YP.. YP

K

M

Fie. 1. Comparison of the amino acid sequences of charybdotoxin (ChTX), iberiotoxin (IbTX), Leiurus Centruvoides quinquestriatus toxin 2 (Lq2), limbatustoxin (LbTX), noxiustoxin (NxTX), margatoxin (MgTX), C. limpidus limpidus toxin I (C.I.I. 1), tityustoxin-Ka (TyKa), agitoxin 1 (AgTXI), agitoxin 2 (AgTX2), agitoxin 3 (AgTX3), kaliotoxin (KTX), and kaliotoxin 2 (KTX2). The sequences have been aligned with respect to the six cysteine residues which are in boldface type. The position of the disulfide bonds is indicated.

Peptidyl inhibitors from scorpion venoms can be subdivided into different groups based on sequence conservation and specificity 2 (Fig. 1). Members of the first group inhibit Ca2+-activated K ÷ channels, although only iberiotoxin (IbTX) and limbatustoxin (LbTX) are specific for the highconductance Ca2+-activated K ÷ channel. All other groups inhibit voltagegated K ÷ channels. However, only members of the Kv1 family are sensitive to inhibition; Kv2, Kv3, and Kv4 type channels are not blocked by these inhibitors. Within the Kvl family, only Kvl.1, 1.2, 1.3, 1.6, and 1.7 are targets of these peptides; Kvl.4 and 1.5 are refractory to inhibition. In this review, we discuss methods for (1) purifying peptides from venom sources, (2) synthesis of the peptides by recombinant techniques, (3) radiolabeling of peptides in biologically active form, and (4) use of radiolabeled peptides to characterize high-affinity receptors in native tissue. Purification of Peptidyl Inhibitors of K ÷ C h a n n e l s from Scorpion Venoms Scorpion venoms contain a vast number of components with activities directed against ion channels. For example, they constitute a rich source

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of peptidyl Na + channel modulators.3 A distinguishing feature of K + channel inhibitors is the existence of a large number of positively charged residues present in these peptides. Thus, it is possible to take advantage of this property to achieve a simple, efficient, and highly reproducible purification procedure. For all peptides that have been purified in our laboratory [charybdotoxin (ChTX), IbTX, LbTX margatoxin (MgTX), agitoxin 1-3 (AgTX1 3)], we have employed two consecutive chromatographic steps: cation-exchange chromatography on a Mono S column (Pharmacia, Piscataway, N J), and reversed-phase chromatography on a C8 or C18 column.4-s This has always afforded the production of pure material as judged by amino acid sequence, amino acid composition, and mass spectroscopic analyses. The first step in peptide purification involves separation of venom components on a Mono S HR5/5 or HR 10/10 column, depending on the amount of material being processed, using a high-performance liquid chromatography (HPLC) system. Lyophilized venom is initially resuspended in 20 mM sodium borate, pH 9.0, at a final protein concentration of ca. 5 mg/ml using vortex agitation. The suspension is then subjected to centrifugation for 15 min at 27,000g at 4° to remove insoluble material. Before applying the soluble material onto the Mono S column, the sample must be filtered through a Millex-GV 0.2-~m pore size filter (Millipore, Bedford, MA) to remove particulate material that could cause obstruction in the HPLC system, thereby increasing back pressure of the column. We do not recommend subjecting the pellet obtained after initial centrifugation to a second extraction. Even after processing the soluble material as described earlier, we frequently observe an increase in the pressure of the system, up to the limit tolerated by the column. If this occurs, the direction of the column must be reversed with continued pumping of buffer until the pressure decreases to normal values. Before injecting sample, the ion-exchange column should be equilibrated with 20 mM sodium borate, pH 9.0, at a flow rate of 0.5 ml/min

3 W. A. Catterall, Ann. Rev. PharmacoL Toxicol. 20, 15 (1980). 4 G. Gimenez-Gallego, M. A. Navia, J. P. Reuben, G. M. Katz, G. J. Kaczorowski, and M. L. Garcia, Proc. Natl. Acad. Sci. U.S.A. 85, 3329 (1988). ~ A. Galvez, G. Gimenez-Gallego, J. P. Reuben, L. Roy-Contancin, P. Feigenbaum, G. J. Kaczorowski, and M. L. Garcia, J. BioL Chem. 265, 11083 (1990). M. Garcia-Calvo, R. J. Leonard, J. Novick, S. P. Stevens, W. Schmalhofer, G. J. Kaczorowski, and M. L. Garcia, J. Biol. Chem. 268, 18866 (1993). 7 M. L. Garcia, M. Garcia-Calvo, P. Hidalgo, A. Lee, and R. MacKinnom Biochemistry 33, 6834 (1994). 8 j. Novick, R. J. Leonard, V. F. King, W. Schmalhofer, G. J. Kaczorowski, and M. L. Garcia, Biophys. J. 59, 78a (1991).

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(HR 5/5 column) or 2 ml/min (HR 10/10 column) depending on the Mono S column employed. The sample is then applied and absorbance monitored at 280 nm. Because of the basic pH conditions, a large amount of material absorbing at 280 nm is not retained by the column and appears in the void volume. We have never observed any biological activity in this material against the K + channels tested and, therefore, it can be discarded. Once the absorbance returns to baseline values, the retained material is eluted with a linear gradient of NaC1 in 20 m M sodium borate, pH 9.0; 0.75 M / h r for H R 5/5 or 0.5 M / h r for H R 10/10 columns. Individual peaks are collected manually and used for determination of their biological activity. These fractions can be stored at - 7 0 ° until further processing is accomplished. The second step of purification is achieved by using a Cls reversedphase H P L C column (25- x 0.46-cm, 5-/zm particle size, or 25- x 1-cm, 5-/zm particle size, depending on the amount of material to be processed; the Separations Group). In some cases, we have also employed a C8 reversed-phase H P L C column and have obtained nearly identical results. The column is equilibrated with 10 m M trifluoroacetic acid (TFA) at a flow rate of either 0.5 ml/min (25- X 0.46-cm column) or 3 ml/min (25- x 1-cm column), depending on the size of the column/amount of material to be loaded. Because most of the peptides are highly charged and do not contain many hydrophobic surfaces in their native conformation, it is very important to have the column well equilibrated in starting buffer. Failure to do this may lead to lack of retention of peptides by the column. Fractions of interest from the Mono S column are then directly applied, without further processing, to the reversed-phase column. Elution is achieved with a linear gradient of organic solvent. We have successfully employed a combination of 2-propanol/acetonitrile (2: 1) in 4 m M TFA, although acetonitrile by itself is also an appropriate solvent for elution. The gradient can be applied from 0 to 40% over either a 30-min period (25- x 0.46-cm column) or a 60-min period (25- x 1-cm column) depending on column size. For best results, it is better to monitor absorbance of eluting material, at least, at two different wavelengths (e.g., 280 and 235 rim). Some peptides have little or no aromatic amino acid content and, therefore, give very small or sometimes undetectable absorbance at 280 nm. Components are separated manually and, in most cases, well-defined peaks with good baseline separation can be obtained. For testing of biological activity, it is suggested that small amounts of material be subjected to lyophilization, and then reconstituted in any buffer containing high ionic strength (e.g., 100 m M NaC1). We also suggest including 0.1% (w/v) bovine serum albumin (BSA) in the resuspension buffer to prevent loss of peptide due to binding to glass or plastic surfaces. Remaining

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material can be stored at - 7 0 °. Once a fraction of interest is identified, and because only small amounts of material are needed for most experiments, it is desirable to lyophilize samples in small aliquots of ca. 20/xg and store them at - 7 0 °. Material stored in this way is usually stable for very long periods of time. When an aliquot is needed, the peptide is resuspended as indicated above and can be stored at 4 ° for several months without loss of biological activity. We do not routinely subject toxin solutions to repetitive freeze-thaw cycles as we have not investigated such procedures with respect to toxin stability. It is important to note that K + channel peptides are typically highly positively charged molecules that will stick to glass surfaces unless a high ionic strength buffer is used to prevent such an interaction from taking place. This is particularly important when drying the peptides in glass tubes. If water is used as the sole resuspension agent, it is likely that most of the peptides will be lost by absorption onto the surface of the tube. Although some material may appear to be chromatographically pure, this does not necessarily mean that it is homogeneous. For example, two different peptide entities may elute together, or a major absorbance peak may contain a component that does not have significant absorbance at the wavelength monitored. To resolve this later situation, it is helpful to monitor absorbance of eluting material at two different wavelengths. If more than one component has eluted together, it is often possible to ascertain that this situation exists by applying the material again to the reversed-phase column, and selecting more shallow gradient elution conditions. Despite all of these precautions, it is still necessary to characterize the material of interest in terms of its amino acid composition, amino acid sequence, and mass spectroscopic properties. When performing automated Edman degradation using most commercially available instruments, it is important to be aware that the last residue of the peptide is usually washed off the filter support, and this amino acid must, therefore, be determined by an independent means. Thus, it is important that the amino acid composition be determined after acid hydrolysis of the peptide, and that it matches the composition obtained by sequence. Furthermore, both of these parameters must correlate well with results obtained by mass spectroscopy. Finally, it is imperative that a peptide of interest be synthesized to confirm that the amino acid sequence determined corresponds to the biological activity of interest. This can be accomplished by either of two independent methods: solid-phase synthesis or biosynthesis by recombinant techniques. This latter approach is discussed in detail in the next section. The solid-phase synthesis of some K + channel inhibitory peptides has been accomplished, and fully reduced peptides have been oxidized to yield material that is indistinguishable from samples purified from crude scorpion

630

TOXINSAND OTHERMEMBRANEACTIVECOMPOUNDS

[32]

venom. 9-I3 This has allowed confirmation of the identities of some peptides, and has further provided a means by which to obtain different variants of these agents for structure-activity relationship studies.

S y n t h e s i s of K + C h a n n e l I n h i b i t o r y Peptides b y Recombinant Techniques The production of K + channel inhibitory peptides by recombinant techniques has become a very popular approach, not only for obtaining large quantities of material at relatively low cost, but also for producing peptide variants with which to carry out mechanistic studies in order to identify those residues important for channel inhibition. 13-16 In general terms, a gene encoding the peptide of interest is inserted into an E s c h e r i c h i a coli expression vector. The peptide is produced as part of a fusion protein, folded, cleaved, and then purified to homogeneity by conventional methods, The first step of this process consists of constructing an artificial gene for the peptide of interest, and inserting it into an appropriate expression vector. We have successfully employed two types of vectors. In pCSP105, the resulting construct encodes a fusion protein of the viral T7 gene 9 product with the K + channel inhibitory peptide, where the two proteins are separated by either a Factor Xa cleavage site, or an enteropeptidase site. In pG9, six histidine residues are inserted between the fusion protein and the Factor Xa cleavage site, at the beginning of the K + channel inhibitory peptide sequence (Fig. 2). This latter construct may facilitate purification of the fusion protein through application of Ni2+-affinity chromatographic techniques. However, in our experience, this type of chromatographic step appears to work only with some constructs, and does not represent a very significant advantage in isolation of the fusion protein. A n appropriate strain of E. coli, such as BL21(DE3), is then transformed 9E. E. Sugg, M. L. Garcia, J. P. Reuben, A. A. Patchett, and G. J. Kaczorowski,J. Biol. Chem. 265, 18745 (1990). 10B. A. Johnson and E. E. Sugg, Biochemistry 31, 8151 (1992). u M. A. Bednarek, R. M. Bugianesi, R. J. Leonard, and J. P. Felix, Biochem. Biophys. Res. Commun. 198, 619 (1994). 12E. Drakopoulou, J. Cotton, H. Virelizier, E. Bernardi, A. R. Schoofs, M. Partiseti, D. Choquet, G. Gurrola, L. D. Possani, and C. Vita, Biochem. Biophys. Res. Commun. 213, 901 (1995). 13j. Aiyar, J. M. Withka, J. P. Rizzi, D. H. Singleton, G. C. Andrews, W. Lin, J. Boyd, D. C. Hanson, M. Simon, B. Dethlefs, C.-L. Lee, J. E. Hall, G. A. Gutman, and K. G. Chandy, Neuron 15, 1169 (1995). 14R. Ranganathan, J. H. Lewis, and R. MacKinnon, Neuron 16, 131 (1996). 15p. Stampe, L. Kolmakova-Partensky,and C. Miller, Biochemistry 33, 443 (1994). 16S. A. N. Goldstein, D. J. Pheasant, and C. Miller, Neuron ll, 1377 (1994).

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SCORWONTOXINSAS TOOLS

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~

631

Peptidetoxin

Ampiciliin resistance

pG9 in

7,8

gene 9 T7 promoter

FI~. 2. Design of the synthetic peptide gene. The plasmid map of K + channel peptides shows the location of the synthetic peptide gene, the Factor Xa cleavage site, the His tag, and T7 gene 9 fusion protein.

with the corresponding plasmid and a single colony is selected to inoculate the culture medium. The cells are grown at 30-37 ° with shaking, and when the optical density of the culture suspension at 650 nm has reached 0.6-0.7, the culture is induced with 0.5 m M isopropylthiogalactoside (IPTG), followed by further incubation for 3 hr at 37 °. Induction of the fusion protein can be assayed by employing 12% S D S - P A G E gels and subsequently staining these with Coomassie blue. After destaining, a major product at ca. 57,000 should be observed on testing the induced culture. If no induction of fusion protein is seen, one should check for proper orientation of the toxin gene, as well as be sure that the cells do not grow above 0.7 optical density units, before addition of IPTG. If induction is successful, cells are pelleted by centifugation and washed once with 50 m M NaC1, 10 m M Tris-HC1, pH 8.0, 1 m M ethylenediaminetetraacetic acid ( E D T A ) , 1 m M dithiothreitol (DTT). Cells are then resuspended in 10 ml/liter of culture using the buffer described earlier. Cells are frozen in liquid N2 and can be stored at - 7 0 ° until further processing. In our experience, one person can easily manage the purification of up to four different peptides at once. For this reason, we store cells at - 7 0 ° until sufficient material is ready for purification. Cells are thawed on ice and the volume is adjusted to 50 ml/ liter of culture with 50 m M NaC1, 10 m M Tris-HC1, p H 8.0, 1 m M E D T A , 1 m M DTT, 100 /xM PMSF (phenylmethylsulfonyl fluoride), 0.5 mg/ml lysozyme. After incubation on ice for 1 hr, the sample is subjected to

632

TOXINS AND OTHER MEMBRANE ACTIVE COMPOUNDS

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sonication four times for 1-min intervals, and then subjected to centrifugation at 27,000g for 15 min. The supernatant is collected and the nucleic acids are removed by precipitation with streptomycin sulfate. It is recommended that at each step, samples be subjected to 12% SDS-PAGE analysis to monitor purification progress. The next step involves purification of the fusion protein by ionexchange chromatography. For this purpose, the supernatant is loaded onto a DEAE-Sepharose fast flow column equilibrated with 50 mM NaC1, 10 mM Tris-HC1, pH 8.0, 1 mM EDTA, 1 mM DTT, 100 /xM PMSF. After extensive washing with equilibration buffer, the fusion protein is batch eluted with 350 mM NaC1, 10 mM Tris-HC1, pH 8.0, 1 mM EDTA, 1 mM DTT, 100/xM PMSF, followed by dialysis overnight at 4° against 100 mM NaC1, 2 0 m M Tris-HC1, pH 8.0, 0.5 mM 2mercaptoethanol. During the dialysis step, folding of the peptide (i.e., formation of disulfide linkages) takes place. Purified fusion protein must undergo enzymatic cleavage in order to release the K + channel inhibitory peptide. To accomplish this, either of two procedures can be employed. In one of these, Factor X, is used to release the peptide from the fusion protein. For this purpose, CaCI2 is added to a final concentration of 3 mM, followed by addition of 1/.~g of Factor Xa per milligram of fusion protein, and the digestion mixture is incubated overnight at room temperature. The time-course of digestion can be followed by SDS-PAGE, but in most cases an overnight incubation is sufficient. We have found this procedure to give reproducible results for given constructs. However, with some peptides, Factor Xa causes a cleavage upstream from its recognition site. We interpret this occurrence as the result of either of two different phenomena: (1) inability of Factor X, to reach its primary site of action (most likely due to steric factors), and/or (2) lack of specificity of Factor Xa. Indeed, if the released peptide containing a part of the fusion protein is purified and subsequently subjected to further incubation with Factor Xa, no further digestion is observed, suggesting that the conformation of the peptide prevents enzyme from reaching its binding site. A different and less expensive way of achieving release of the K +channel inhibitory peptide from its fusion protein is through the use of trypsin. For this purpose, CaC12 is added to a final concentration of 5 mM, and sample is incubated with 5/zg trypsin per milligram of fusion protein for 30 min at room temperature. In this case, time of incubation with enzyme is critical since longer incubations could lead to cleavage within the peptide. This procedure, however, works well with all constructs tested so far, and it is less expensive than those protocols involving Factor X~. Even in those situations where Factor Xa produced wrong cleavage of the fusion protein,

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633

it is possible to treat the resulting sample with trypsin to release the appropriate peptide. The K + channel inhibitory peptides must next be purified from the rest of the digestion mixture. This can be accomplished, as described in the previous section, by loading the sample onto a Mono S HR 10/10 column equilibrated with 20 mM sodium borate, pH 9.0. The fusion protein and various proteolytic fragments are not retained by the column under these conditions. Highly positively charged peptides, such as ChTX, MgTX, noxiustoxin (NxTX), and AgTX~ 3, can be loaded directly onto the column, whereas less positively charged peptides, such as IbTX, must first be dialyzed against 20 mM sodium borate, pH 9.0, before loading. Bound peptides are eluted in the presence of a linear gradient of NaC1 in 20 mM sodium borate, pH 9.0 (0.5 M/hr) at a flow rate of 2 ml/min. A major absorbance peak representing the peptide of interest is usually observed. Certain peptides, such as ChTX and IbTX, have their amino-terminal group cyclized in the form of pyroglutamine. Because cyclization is important for the biological activity of these peptides, this reaction must be accomplished before the last purification step. For this purpose, HPLC-grade acetic acid is added to the Mono S sample at a final concentration of 5%, and the mixture is incubated at either 65° (ChTX) or 45° (IbTX) until full cyclization of the N-terminal amino group is achieved. Cyclization is followed by N-terminal sequencing, since the peptide species with the blocked N-terminal is resistant to Edman degradation. For ChTX, cyclization takes place during an overnight incubation with acetic acid, whereas for IbTX, this process may take up to 2 days, perhaps due to the lower incubation temperature used. We have found that at higher temperatures, IbTX is not stable in the presence of acetic acid, as indicated by the appearance of different chromatographic species with time. The final stage of peptide purification is achieved on reversed-phase chromatography, using a semipreparative C~8 reversed-phase HPLC column, employing the same conditions as those described in the previous section. The composition of the purified peptide should be checked by N-terminal sequencing, amino acid hydrolysis, and/or mass spectroscopic analysis. Production of peptides through recombinant techniques is an easy way of obtaining large quantities of material at low cost. The yields of purified peptide per liter of E. coli culture vary depending on the construct, but it is highly reproducible for any given peptide. For instance, 10-15 mg of MgTX per liter of culture can routinely be obtained, whereas for ChTX, the yields are lower: 1-5 mg of peptide per liter of culture. We believe that these differences in yield are related to the efficiency of the cleavage step since amounts of purified fusion protein produced are similar in all cases.

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TOXINS AND OTHER MEMBRANE ACTIVE COMPOUNDS

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Radiolabeling of K ÷ C h a n n e l I n h i b i t o r y Peptides The goal for radiolabeling K + channel inhibitory peptides is to obtain a biologically active derivative that can be used to identify the target protein in tissues of interest, and further to develop the molecular pharmacology of given K + channels. Radiolabeled peptides at high specific activity can be produced after reaction of peptidyl Tyr or His residues with Na125I. Alternatively, Lys residues can be covalently modified by reaction with leSI-labeled B o l t o n - H u n t e r reagent. Because certain Lys residues in K + channel inhibitory peptides are crucial for their activity, 16,17 this latter m e t h o d has not been successfully employed to date. Labeling of Tyr residues in C h T X and M g T X has been successful and, thus, these radiolabeled peptides have played a crucial role in characterizing K + channels] 8,19 O t h e r peptides such as AgTX1_3 do not contain Tyr residues, or, as in the case of IbTX, iodirlation of the Tyr residue leads to complete loss of biological activity. In these situations, it is possible to engineer amino acid residues into the sequence so that they can be subsequently modified by incorporation of a radiolabeled tag at a position that is not critical for biological activity, e°-23 Iodination of peptides such as ChTX, MgTX, and I b T X - D 1 9 Y / Y 3 6 F has been achieved using either of two methods: the I o d o - G e n (Pierce, Rockford, IL) or glucose oxidase/lactoperoxidase protocols. The latter utilizes beads to which the enzymes have been immobilized. Use of this system usually yields chromatograms displaying fewer reaction side products, due to the milder oxidizing condition of the procedure. Unfortunately, the manufacturer of these beads, Bio-Rad (Richmond, CA), has discontinued their production. In the reaction with I o d o - G e n , the water-insoluble reagent is immobilized on the surface of a glass vial. A solution of peptide and Na125I is added, and after a certain period of time, the reaction mixture is r e m o v e d and injected onto a reversed-phase H P L C column for purification of the radiolabeled peptide. For a typical iodination, a solution of I o d o - G e n reagent is p r e p a r e d in acetone and an aliquot corresponding to 0.5-1.0/xg of reagent is placed at the b o t t o m of a vial and dried under argon or nitrogen. Once dried, the ~7C.-S. Park, and C. Miller, Neuron 9, 307 (1992). 18j. Vazquez, P. Feigenbaum, G. Katz, V. F. King, J. P. Reuben, L. Roy-Contancin, R. S. Slaughter, G. J. Kaczorowski, and M. L. Garcia, J. Biol. Chem. 264, 20902 (1989). 19H.-G. Knaus, R. O. A. Koch, A. Eberhart, G. J. Kaczorowski, M. L. Garcia, and R. S. Slaughter, Biochemistry 34, 13627 (1995). 2oS. K. Aggarwal and R. MacKinnon, Neuron 16, 1169 (1996). 21H.-G. Knaus, C. Schwarzer, R. O. A. Koch, A. Eberhart, G. J. Kaczorowski,H. Glossmann, F. Wunder, O. Pongs, M. L. Garcia, and G. Sperk, J. Neurosci. 16, 955 (1996). 22E. Shimony, T. Sun, L. Kolmakova-Partensky, and C. Miller, Prot. Eng. 7, 503 (1994). z3A. Koschak, R. O. Koch, J. Liu, G. J. Kaczorowski, P. H. Reinhart, M. L. Garcia, and H.-G. Knaus, Biochemistry 36, 1943 (1997).

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vial can be rinsed several times with buffer to remove any aqueous soluble contaminants. Then, a solution containing 10-20/~g of peptide in 100 m M sodium phosphate, p H 7.3-8.0, is added, followed by addition of 2 - 5 / x C i of Na125I (2200 Ci/mmol). After mixing, the vial is capped and the reaction is allowed to proceed at room temperature for 10-15 min. One minute before the end of the reaction, the vial is open to the air, the iodination mixture is removed with a syringe, and it is injected onto a C~8 reversedphase H P L C column that has been equilibrated with 10 m M TFA. All of these procedures should be carried out inside a hood with appropriate ventilation; special care should be taken when loading the iodination mixture onto the H P L C column because the low p H buffer employed could lead to the generation of radioactive iodine gas, which will permeate skin and accumulate in the thyroid gland. For this reason, the loading and washing volumes from the H P L C column are always collected in 1 N N a O H to block formation of iodine gas. H P L C column elution conditions will depend on the peptide under investigation, and it is recommended that the iodination reaction be performed with unlabeled NaI first, in order to optimize the separation conditions. With ChTX, for instance, a linear gradient of 2-propanol/acetonitrile 2:1 (5-14%, 40 min) at a flow rate of 0.5 ml/min leads to a well-defined and well-separated peak corresponding to monoiodotyrosine ChTX, based on the specific activity of the radiolabeled peptide and on sequence analysis of the modified toxin. Because the amounts of material used in these procedures is low, changes in absorbance should be monitored at 210 nm. To store radiolabeled peptides for extended periods of time, the fraction of interest is made 0.1% (w/v) in BSA, lyophilized, and reconstituted with 100 m M NaC1, 20 m M Tris-HC1, pH 7.4. Aliquots of this material containing ca. 5/zCi are frozen in liquid N: and stored at - 7 0 °. When needed, fractions are thawed and then stored at 4 °. These handling conditions have been found to be optimal in that no loss of biological activity of the labeled peptide has been observed, even after several months of storage. In most cases, in addition to the monoiodotyrosine derivative, a second minor peak corresponding to the diiodotyrosinepeptide is obtained. We usually do not retain this material since in many cases its biological activity is undefined. An alternative method for introducing a radiolabel into these peptides makes use of an analog in which a Cys residue has been placed at a position in the peptide that is not critical for bioactivity. The free sulfydryl is then reacted with [3H]N-ethyl-maleimide (NEM) to produce material with full biological activity, but at lower specific activity than that obtained with NalZSI labeling (e.g., ca. 60 Ci/mmol). This procedure has been successfully employed to radiolabel ChTX and IbTX making use of position 19, 2a,23 and with AgTX~ and AgTX2 at position 20. 20 Note that this type of reaction is not restricted solely to radiolabeling of the peptide; other fluorescent and

636

TOXINS AND OTHER

MEMBRANE

[32]

ACTIVE COMPOUNDS

-2.0

-2.0

.2.0

c

1 o

L

- 1.0

/•-I.0

2'5 3'0 Time (rain)

3'5

ol

~-o

-0

a'0

-1.0

2'0

2'5 30 Time (min)

3'5

2'0

2'5

3'0

Time (rnin)

FZG. 3. Synthesis of [3H]IbTX-D19C-NEM. An aliquot of IbTX-D19C in 50 mM sodium phosphate, pH 7.0, was treated with 2 mM (A) or 10 mM (B) DTT for 1 hr at room temperature. Peaks 1 and 2 in (A) with retention times of 29.8 and 30.2 min, respectively, represent the unreduced and reduced forms at position 19 of IbTX-D19C. In (B) the retention time of the reduced peptide is 30.3 min. (C) Reduced IbTX-D19C prepared as in (B) was reacted with 1 mCi [3H]NEM (56 Ci/mmol) for 1 hr at 37°. Samples were loaded onto a C18 reversedphase column equilibrated with 10 mM TFA. Elution was achieved in the presence of a linear gradient (7-25%, 30 min) of 2-propanol/acetonitrile (2:1) in 4 mM TFA. Peaks 1 and 2 with retention times of 27.5 and 28.9 rain, respectively, have identical specific activity and their biological activity is also indistinguishable. biotinylated derivatives of N E M can also be reacted to yield interesting peptidyl ion channel probes. H o w e v e r , there is one consideration to note with this a p p r o a c h which concerns the fact that a newly i n t r o d u c e d Cys residue is not initially reactive on purification of the peptide, since it does not exist as a free sulfhydryl group. Instead, it exists in a disulfide bridge with either 2 - m e r c a p t o e t h a n o l or a n o t h e r toxin molecule. It is necessary, therefore, to selectively r e d u c e this disulfide b o n d without disrupting the integrity of the natural disulfide bridges of the peptide that are crucial for biological activity before labeling with the sulfhydryl reagent. T o accomplish this, peptides are incubated in 50 m M sodium phosphate, p H 7.0, with 1 - 1 0 m M D T T at r o o m t e m p e r a t u r e for 1 hr. T h e reaction mixture is then applied to a C~8 reversed-phase H P L C column. Elution conditions must be optimized for each particular peptide preparation. T h e peptide containing the free-sulfhydryl g r o u p should be easily identified by a change in retention time w h e n c o m p a r e d with u n r e d u c e d material (Fig. 3A). T h e change in retention time m a y not be very large and, therefore, a p p r o p r i a t e elution conditions should be used to achieve g o o d separation. In the event that the separation is suboptimal, this should not preclude continuation of the p r o c e d u r e since u n r e d u c e d material will not react with N E M . In o u r experience, optimal r e c o v e r y of C 1 9 / C 2 0 r e d u c e d peptide approximates 50%. A l t h o u g h higher concentrations of either D T T or increases in p H can lead to full disappearance of starting material (Fig. 3B), they also cause

[32]

SCORPIONTOXINSAS TOOLS

637

appearance of other forms of the peptide with much longer retention times in which additional disulfide bonds have been reduced. This material displays no biological activity and can be discarded. Reduced peptide can be concentrated to about 20/xl using a Speed-Vac, and then 100/xl of 50 m M sodium phosphate is added. It is important to use reduced peptide for reaction with [3H]NEM as soon as possible after its production. We have observed that, in some cases, reduced peptide can dimerize via formation of a disulfide linkage, even when stored at - 7 0 °. For the alkylating reaction, one vial containing 1 mCi of [3H]NEM in pentane is opened and the content transferred to a glass tube. The solvent is evaporated under N2 to about 300 txl, and then 200/xl of 50 m M sodium phosphate, p H 7.0, is added. After mixing by vortex, the remaining pentane is evaporated, the [3H]NEM solution is added to the peptide solution, and the vial is rinsed with an additional 200/xl of 50 m M sodium phosphate, p H 7.0, which is then also transferred to the peptide solution. The reaction mixture is allowed to react for 1 hr at 37 °, and then injected onto a Cls reversed-phase H P L C column equilibrated with 10 m M TFA. Elution conditions should be optimized for each individual peptide, and alkylated material can be easily identified by the change in retention time. In addition, radioactivity should be associated with this material. If good separation is achieved, the specific activity of the alkylated peptide should correspond to that of the [3H]NEM employed for the reaction, ca. 60 Ci/mmol. It is worth noting that in the case of reaction of IbTX-C19 with [3H]NEM, two well-separated radiolabeled peaks are obtained in equivalent amounts, and both display identical biological activity (Fig. 3C). The exact nature of these two species is unknown, but this may correspond to the production of two isomers of IbTX-C19-NEM.

R e c e p t o r Binding S t u d i e s The goal of radiolabeling a given K + channel inhibitory peptide is so that it can be used to characterize its receptor in native tissues. This includes characterizing the kinetics of ligand association and dissociation, the molecular pharmacology of the channel, and the tissue distribution of the channel] s,19,23-26 Additionally, radiolabeled peptides have been successfully em24j. Vazquez, P. Feigenbaum, V. F. King, G. J. Kaczorowski,and M. L. Garcia, J. Biol. Chem. 265, 15564 (1990). 25H.-G. Knaus, O. B. McManus, S. H. Lee, W. A. Schmalhofer, M. Garcia-Calvo, L. M. H. Helms, M. Sanchez, K. Giangiacomo, J. P. Reuben, A. B. Smith III, G. J. Kaczorowski, and M. L. Garcia, Biochemistry 33, 5819 (1994). 26O. B. McManus, G. H. Harris, K. M. Giangiacomo, P. Feigenbaum, J. P. Reuben, M. E. Addy, J. F. Burka, G. J. Kaczorowski, and M. L. Garcia, Biochemistry 32, 6128 (1993).

638

TOXINSAND OTHER MEMBRANEACTIVECOMPOUNDS

[321

ployed in the purification of native ion channels. 27-3° As a matter of fact, all auxiliary (/3) subunits of ion channels have been identified and subsequently characterized through the purification of the corresponding ion channel complex using binding of channel probes as a monitor of channel purification. There are different ways in which to monitor p e p t i d e - K + channel interactions, such as autoradiographic techniques or binding of ligand to intact cells or tissues, but perhaps the most c o m m o n studies are those in which the peptide interaction is measured in highly enriched m e m b r a n e preparations. T h r e e major considerations should be taken into account when considering the development of a binding reaction involving K ÷ channel inhibitory peptides discussed in this chapter. The first one concerns the potencies of the peptides as inhibitors of K + channels; under physiologic conditions, some of these peptides display n a n o m o l a r affinities, but their potency can be greatly enhanced by selecting appropriate binding reaction conditions (i.e., by lowering ionic strength of the incubation medium, which will enhance electrostatic interaction between positively charged residues on the peptide and negatively charged residues in the channel's vestibule? 1 33 Second, problems arising from the handling of these peptides should be considered. The easiest way to separate bound from free ligand is by using filtration techniques and, therefore, the filters used must have specific characteristics, or be treated accordingly to minimize binding of the positively charged peptides. We have found that G F / C glass fiber filters (Whatman, England) presoaked in 0.5-1.0% (w/v) polyethyleneimine (Sigma, St. Louis, M O ) provide a low background binding support if high ionic strength buffer (e.g., 100 m M NaC1, 20 m M Tris-HC1, p H 7.4) is used to quench the binding reaction. This high ionic strength quench buffer is also important to induce immediate dissociation of toxin bound to low-affinity sites. We should also consider the physical properties of the test tubes in which the binding reaction is carried out. If low ionic strength media are used, then glass tubes should be avoided because peptides may adhere to these surfaces; under such conditions, use of polystyrene tubes is recommended. In addition, inclusion of 0.1% (w/v) BSA in the incubation medium would also 27M. Garcia-Calvo, H.-G. Knaus, O. B. McManus, K. M. Giangiacomo, G. J. Kaczorowski, and M. L. Garcia, J. Biol. Chem. 269, 676 (1994). 2s K. M. Giangiacomo, M. Garcia-Calvo, H.-G. Knaus, T. J. Mullmann, M. L. Garcia, and O. McManus, Biochemistry 34, 15849 (1995). 29D. N. Parcej and J. O. Dolly, Biochem. J. 25'7, 899 (1989). 3oH. Rehm, and M. Lazdunski, Proc. Natl. Acad. Sci. U.S.A. 85, 4919 (1988). 31K. M. Giangiacomo, M. L. Garcia, and O. B. McManus, Biochemistry 31, 6719 (1992). 32R. MacKinnon and C. Miller, J. Gen. Physiol. 91, 335 (1988). 33S. Candia, M. L. Garcia, and R. Latorre, Biophys. J. 63, 583 (1992).

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prevent peptide from being absorbed onto the surface of tubes. Finally, the last consideration is the source of receptor. For instance, C h T X blocks two different types of K + channels: high-conductance Ca2+-activated K + channels, and voltage-dependent K + channels. If one considers using this peptide as a ligand, it is important to select a tissue in which either, but not both, of these channels is present. Moreover, use of highly enriched plasma m e m b r a n e preparation should give a higher ratio of specific to nonspecific binding and should be employed if at all feasible. Other parameters which are optimal for each particular ligand-receptor interaction should be determined according to each individual situation. For example, the incubation time necessary to reach equilibrium for any particular ligand can vary considerably from about 1 hr for [~2SI]MgTX or [12sI]ChTX]9'24 to close to 60-72 hr for [125I]IbTX.23 Summary The search for peptidyl inhibitors of K + channels is a very active area of investigation. In addition to scorpion venoms, other v e n o m sources have been investigated; all of these sources have yielded novel peptides with interesting properties. For instance, spider venoms have provided peptides that block other families of K + channels (e.g., Kv2 and K v 4 ) that act via mechanisms which modify the gating properties of these channels. 34-36 Such inhibitors bind to a receptor on the channel that is different from the pore region in which the peptides discussed in this chapter b i n d s In fact, it is possible to have a channel occupied simultaneously by both inhibitor types. It is expected that many of the methodologies concerning peptidyl inhibitors from scorpion venom, which have been developed in the past and outlined above, will be extended to the new families of K + channel blockers currently under development.

34M. C. Sanguinetti, J. H. Johnson, L. G. Hammerland, P. R. Kelbaugh, R. A. Volkmann, N. A. Saccomano, and A. L. Mueller, Molec. Pharmacol. 51, 491 (1997). 35K. J. Swartz and R. MacKinnon, Neuron 15, 941 (1995). 36K. J. Swartz and R. MacKinnon, Neuron 18, 665 (1997). 37K. J. Swartz and R. MacKinnon, Neuron 18, 675 (1997).