Stress (Takotsubo) Cardiomyopathy During Liver Transplantation: Case Study and Literature Review.

A case of stress (takotsubo) cardiomyopathy (TC) that occurred intraoperatively during liver transplantation surgery was identified by transesophageal echocardiography. Only a few cases of TC occurring during liver transplantation have been reported to date. Unlike other cases reported, TC occurred during the anhepatic stage of the liver transplantation, with subsequent complete recovery. Notwithstanding the large number of cases of TC in the perioperative settings reported worldwide, the exact reasons of this syndrome occurring intraoperatively as well as precipitating factors and conditions remain mostly unknown.

Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor.

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Lifetime cost effectiveness of different brands of prosthesis used for total hip arthroplasty: a study using the NJR dataset.

There is little evidence on the cost effectiveness of different brands of hip prostheses. We compared lifetime cost effectiveness of frequently used brands within types of prosthesis including cemented (Exeter V40 Contemporary, Exeter V40 Duration and Exeter V40 Elite Plus Ogee), cementless (Corail Pinnacle, Accolade Trident, and Taperloc Exceed) and hybrid (Exeter V40 Trilogy, Exeter V40 Trident, and CPT Trilogy). We used data from three linked English national databases to estimate the lifetime risk of revision, quality-adjusted life years (QALYs) and cost. For women with osteoarthritis aged 70 years, the Exeter V40 Elite Plus Ogee had the lowest risk of revision (5.9% revision risk, 9.0 QALYs) and the CPT Trilogy had the highest QALYs (10.9% revision risk, 9.3 QALYs). Compared with the Corail Pinnacle (9.3% revision risk, 9.22 QALYs), the most commonly used brand, and assuming a willingness-to-pay of £20,000 per QALY gain, the CPT Trilogy is most cost effective, with an incremental net monetary benefit of £876. Differences in cost effectiveness between the hybrid CPT Trilogy and Exeter V40 Trident and the cementless Corail Pinnacle and Taperloc Exceed were small, and a cautious interpretation is required, given the limitations of the available information. However, it is unlikely that cemented brands are among the most cost effective. Similar patterns of results were observed for men and other ages. The gain in quality of life after total hip arthroplasty, rather than the risk of revision, was the main driver of cost effectiveness. Cite this article: Bone Joint J 2015;97-B:762-70.

Switch and template pattern formation in a discrete reaction-diffusion system inspired by the Drosophila eye.

We examine a spatially discrete reaction-diffusion model based on the interactions that create a periodic pattern in the Drosophila eye imaginal disc. This model is known to be capable of generating a regular hexagonal pattern of gene expression behind a moving front, as observed in the fly system. In order to better understand the novel "switch and template" mechanism behind this pattern formation, we present here a detailed study of the model's behavior in one dimension, using a combination of analytic methods and numerical searches of parameter space. We find that patterns are created robustly, provided that there is an appropriate separation of timescales and that self-activation is sufficiently strong, and we derive expressions in this limit for the front speed and the pattern wavelength. Moving fronts in pattern-forming systems near an initial linear instability generically select a unique pattern, but our model operates in a strongly nonlinear regime where the final pattern depends on the initial conditions as well as on parameter values. Our work highlights the important role that cellularization and cell-autonomous feedback can play in biological pattern formation.

Evaluation of the cost-effectiveness of root canal treatment using conventional approaches versus replacement with an implant.

AIM: To evaluate the cost-effectiveness of root canal treatment for a maxillary incisor tooth with a pulp infection, in comparison with extraction and replacement with a bridge, denture or implant supported restoration. METHODOLOGY: A Markov model was built to simulate the lifetime path of restorations placed on the maxillary incisor following the initial treatment decision. It was assumed that the goal of treatment was the preservation of a fixed platform support for a crown without involving the adjacent teeth. Consequently, the model estimates the lifetime costs and the total longevity of tooth and implant supported crowns at the maxillary incisor site. The model considers the initial treatment decisions, and the various subsequent treatment decisions that might be taken if initial restorations fail. RESULTS: Root canal treatment extended the life of the tooth at an additional cost of pound5-8 per year of tooth life. Provision of orthograde re-treatment, if the root canal treatment fails returns further extension of the expected life of the tooth at a cost of pound12-15 per year. Surgical re-treatment is not cost-effective; it is cheaper, per year, to extend the life of the crown by replacement with a single implant restoration if orthograde endodontic treatment fails. CONCLUSION: Modelling the available clinical and cost data indicates that, root canal treatment is highly cost-effective as a first line intervention. Orthograde re-treatment is also cost-effective, if a root treatment subsequently fails, but surgical re-treatment is not. Implants may have a role as a third line intervention if re-treatment fails.

Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg, approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.

Designed peptide analogues of the potassium channel blocker ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.

Thirteen UDPglucuronosyltransferase genes are encoded at the human UGT1 gene complex locus.

The original novel UGT1 complex locus previously shown to encode six different UDP-glucuronosyltransferase (transferase) genes has been extended and demonstrated to specify a total of 13 isoforms. The genes are designated UGT1A1 through UGT1A13p with four pseudo ones. UGT1A2p and UGT1A11p through UGT1A13p have either nucleotide deletions or flawed TATA boxes and are therefore pseudo. In the 5' region of the locus, the 13 unique exons 1 are arranged in a tandem array with each having its own proximal TATA box element and, in turn, are linked to four common exons to allow for the independent transcriptional initiation to generate overlapping primary transcripts. Only the lead exon in the nine viable primary transcripts is predicted to undergo splicing to the four common exons generating mRNAs with identical 3' ends and transferase isozymes with an identical carboxyl terminus. The unique amino terminus specifies acceptor-substrate selection, and the common carboxyl terminus apparently specifies the interaction with the common donor substrate, UDP-glucuronic acid. In the extended region, the viable TATA boxes are either A(A)TgA(AA)T or AT14AT; in the original locus the element for UGT1A1 is A(TA)7A and TAATT/CAA(A) for all of the other genes. UGT1A1 specifies the critically important bilirubin transferase isoform. The relationships of the exons 1 to each other are as follows: UGT1A2p through UGT1A5 comprises a cluster A that is 87-92% identical, and UGT1A7 through UGT1A13p comprises a cluster B that is 67-91% identical. For the two not included in a cluster, UGT1A1 is more identical to cluster A at 60-63%, whereas UGT1A6 is identical by between 48% and 56% to all other unique exons. The locus was expanded from 95 kb to 218 kb. Extensive probing of clones beyond 218 kb with coding nucleotides for a highly conserved amino acid sequence present in all transferases was unable to detect other exons 1. The mRNAs are differentially expressed in hepatic and extrahepatic tissues. This locus is indeed novel, indicating the least usage of exon sequences in specifying different transferase isozymes that have an expansive substrate range.

A helical capping motif in ShK toxin and its role in helix stabilization.

ShK toxin, a 35-residue polypeptide cross-linked by three disulfides, is a potent blocker of voltage-gated potassium channels and is of interest as a lead in the development of new immunosuppressant agents. ShK toxin contains two short stretches of alpha-helix, the first of which is preceded by a putative N-capping box encompassing residues Thr13 and Gln16. (1)H and (13)C NMR data support the presence of this structural motif, but the hydrogen bonds involving residues 13 and 16 in the solution structure of ShK toxin do not match the pattern expected for a conventional N-cap motif. They do, however, fit the pattern for the recently described ST-motif, class 4a (Wan and Milner-White (1999) Journal of Molecular Biology, 1999, Vol. 286, pp. 1651-1662). The (1)H NMR chemical shifts, nuclear Overhauser effects, and amide exchange rates of native ShK toxin are compared with those of three synthetic analogues with the substitutions Thr13 to Ala and Gln16 to Glu and Ala in order to determine the contribution of this motif to the structure and stability of ShK toxin. Disruption of the capping interactions destabilizes the helices, with the Thr13 to Ala substitution being much more disruptive than Gln16 to Ala, consistent with the lack of hydrogen bonding to the side chain of residue i + 4 in a class 4a ST-motif. Mutation of residues 13 and 16 has only a minor effect on potassium channel binding, probably because the disulfide bonding network minimizes the effect of loss of the capping motif on the overall structure. The implications of these findings for the design of ShK analogues are discussed.

Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels.

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca(2+)-activated K(+) channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca(2+)-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys(32), which lies in close proximity to this cluster. These analogs block IKCa1 with approximately 20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1. 6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K(Ca) channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K(+) channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K(+) channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.

Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35 residue polypeptide cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. To investigate the role of these disulfides in the structure and channel-blocking activity of ShK toxin, a series of analogues was synthesized by selective replacement of each pair of half-cystines with two alpha-amino-butyrate (Abu) residues. The remaining two disulfide pairs were formed unambiguously using an orthogonal protecting group strategy of Cys(Trt) or Cys(Acm) at the appropriate position. The peptides were tested in vitro for their ability to block Kv1.1 and Kv1.3 potassium channels and their ability to displace [(125)I]dendrotoxin binding to rat brain synaptosomal membranes. The monocyclic peptides showed no activity in these assays. Of the dicyclic peptides, [Abu12,28]ShK(3-35,17)(-)(32) (where the subscript indicates disulfide connectivities) had weak activity on Kv1.3 and Kv1.1. [Abu17,32]ShK(3-35,12)(-)(28) blocked Kv1.3 with low nanomolar potency, but was less effective (being comparable to [Abu12,28]ShK(3-35,17)(-)(32)) against Kv1.1. [Abu3, 35]ShK(12-28,17)(-)(32), retained high picomolar affinity against both channels. Corroborating these results, [Abu3,35]ShK(12-28, 17)(-)(32) had an IC(50) ratio relative to native toxin of 18 in the displacement assay, whereas [Abu17,32]ShK(3-35,12)(-)(28) and [Abu12, 28]ShK(3-35,17)(-)(32) had ratios of 69 and 390, respectively. Thus, the disulfide bond linking the N- and C-terminal regions is less important for activity than the internal disulfides. NMR analysis of the [Abu12,28] and [Abu17,32] analogues indicated that they had little residual structure, consistent with their significantly reduced activities. By contrast, [Abu3,35]ShK(12-28,17)(-)(32) had a moderately well-defined solution structure, with a mean pairwise root-mean-square deviation of 1.33 A over the backbone heavy atoms. This structure nevertheless showed significant differences from that of native ShK toxin. The possible interactions of this analogue with the channel and the distinction between native secondary and tertiary structure on one hand and global topology imposed by the disulfide bridges on the other are discussed.

Comparison of inhibitor binding to feline and human immunodeficiency virus proteases: structure-based drug design and the resistance problem.

The design and synthesis of compounds targeted against human immunodeficiency virus 1 (HIV-1) protease have resulted in effective antiviral therapies. However, the rapid replication of the virus and the inherent mutability of the viral genome result in the outgrowth of resistant strains in the majority of patients. Thus, there is a continuing need to develop new antiprotease compounds that may bind more effectively to the resistant forms of protease. This contribution examines the binding of a single inhibitor to two different retroviral proteases, HIV-1 protease and feline immunodeficiency virus protease. Despite the overall similarity of the related retroviral enzymes, specific substitutions within the binding site cavity provide a distinctly different binding landscape that dramatically alters the affinity of compounds. Through this comparison, insights have been obtained into new strategies for drug design. New compounds based on these concepts have been tested against the two enzymes.

Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.

ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide.

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.

Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin.

The effects of pH, temperature and polypeptide concentration on the solution structure and side chain interactions of ShK toxin, a potassium-channel-blocking polypeptide from the sea anemone Stichodactyla helianthus, have been investigated by means of one-dimensional and two-dimensional 1H-NMR spectroscopy. Resonance assignments have been obtained for most protons in the molecule, and for the alpha and beta carbon atoms. The lack of concentration dependence of the 1H chemical shifts and linewidths indicates that self-association is not significant and cannot account for the sheet-like structure near the N terminus. The structure is stable to high temperature, showing little change even at 353 K. This stability allowed backbone-amide temperature coefficients to be interpreted, and the correlation of these values with hydrogen bonds observed in the structures and with solvent exchange rates is discussed. pKa values have been measured for Asp5, His19 and Tyr23, and the contributions to these pKa values from other residues investigated using the analogues R11Q (denoting substitution of Argll with Gln), R11E, H19K, K22A, Y23A and K30A. These results show that Asp5 (pKa 2.8) makes an electrostatic interaction with Lys30, which may be partially responsible for the importance of these side chains in the folding of synthetic toxin. The phenolic pKa of Tyr23 is reduced to 8.7 in the native toxin, as a result of interactions with the positively charged side chains of Arg11 and to a lesser extent Lys22. Several hydrogen bonds between the Arg11 guanidino group and the Tyr23 phenolic group are found in the solution structures. As these three residues are implicated in the tight binding of ShK toxin to the T-lymphocyte voltage-gated potassium channel Kv1.3, their close interactions should be taken into account in models of binding of this toxin to the pore and vestibule of this and other potassium channels.

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8).

A genomic clone encoding the protease (Pr) and the assembly protein (AP) of Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) has been isolated and sequenced. As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame. The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively. The amino acid sequence of KSHV Pr has 56% identity with that of herpesvirus salmiri, the most similar virus by phylogenetic comparison. Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells. Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity. Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme. The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu-Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family. The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion. These features of the viral protease provide opportunities to develop specific inhibitors of its enzymatic activity.

An essential binding surface for ShK toxin interaction with rat brain potassium channels.

An "Ala scan" analysis of ShK toxin, a 35-residue basic peptide possessing three disulfide bonds, identifies seven side chains which influence binding to brain delayed rectifier potassium channels. Additional analogs were synthesized and tested to further decipher the roles of these residues, particularly Tyr23. The inhibitory effects of these analogs on 125I-labeled dendrotoxin binding to rat brain membranes showed that replacement of Tyr23 with Ala drastically lowered the affinity of the toxin for the Kv1.2 channels. Ala substitution of Phe27 reduced potency more than 15-fold. Monosubstituted Ala analogs for Ile7, Ser20, or Lys30 each displayed 5-fold reductions in potency. Thus, aromaticity at position 23 is important for effective delayed rectifier brain K channel binding. In contrast, the aromatic residue at position 27 was not critical, since cyclohexylalanine substitution increased affinity. The solution structure of ShK toxin clusters Ile7, Arg11, Ser20, Lys22, Tyr23, and Phe27 in close proximity, forming the potassium channel binding surface of the toxin. We propose an essential binding surface on the toxin in which Lys22 and Tyr23 are major contributors, through ionic and aromatic (hydrophobic) interactions, with the potassium channel.

Identification of three separate binding sites on SHK toxin, a potent inhibitor of voltage-dependent potassium channels in human T-lymphocytes and rat brain.

Eighteen synthetic analogs of ShK toxin, a thirty-five residue K channel blocker derived from the sea anemone Stichodactyla helianthus, were prepared in order to identify functionally important residues. CD spectra of sixteen of the analogs were virtually identical with the spectrum of wild-type toxin, indicating that the conformations were not affected by the substitutions. A conserved residue, Lys22, is essential for ShK binding to rat brain K channels which are primarily of the Kv1.2 type. However, a cationic side chain at position 22 is not essential for binding to the human Jurkat T-lymphocyte Kv1.3 channel. While decreasing bulkiness at this position affected toxin affinity for the brain K channels, increasing bulkiness decreased toxin affinity for both brain and lymphocyte K channels. In contrast to the rat brain channels, ShK binding to Kv1.3 was sensitive to substitution at Lys9 and Arg11.

Synthesis and structural characterisation of analogues of the potassium channel blocker charybdotoxin.

Charybdotoxin is a 37-residue polypeptide toxin from scorpion venom, which acts by blocking voltage-gated and Ca(2+)-activated K+ channels. We have synthesized charybdotoxin and three mono-substituted analogues using an Fmoc-tBu protocol. The Phe-2 --> Tyr analogues was chosen to introduce a site for Tyr iodination which was distinct from the K+ channel binding surface, while the Glu-12 --> Gln and Arg-19 --> His analogues were studied to probe the roles of charged residues at these positions in the structure and activity of the toxin. The synthetic native molecule was equipped with natural toxin in inhibiting the human erythrocyte Ca(2+)-dependent K+ channel. The affinities of all three analogues for the erythrocyte K+ channel were slightly reduced, with the Arg-19 --> His analogue showing the greatest increase in IC50 (2.30-fold). Two-dimensional 1H-NMR studies of these analogues showed that the Glu-12 to Gln substitution, which appeared to destabilise the N-terminal half of the alpha-helix, possibly due to the weakening of an N-terminal helix capping interaction which is apparent from our NMR data. His-21 has a pKa more than one unit below the value for a non-interacting histidine. Possible reasons for this are that the imidazolium side chain is partly buried and is located near positively charged moieties. Thus, His-21 would be neutral at physiological pH, where charybdotoxin binds to the potassium channel.