Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Na_v1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Na_v1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Na_v1.4. Here, we determine the NMR structure of a selectively ¹³C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By ¹³C NMR, we obtain NMR structural constraints as ¹³C chemical shifts and the ¹H-²H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the ¹H-²H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel Na_vRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Na_v1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.     

Neuronally micro-conotoxins from Conus striatus utilize an alpha-helical motif to target mammalian sodium channels

Mu-conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of mu-conotoxins SIIIA and SIIIB by (125)I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced (125)I-TIIIA from native rat brain Na(v)1.2 (IC(50) values 10 and 5 nm, respectively) and muscle Na(v)1.4 (IC(50) values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Na(v)1.2 and Na(v)1.4. An alanine scan of SIIIA-(2-20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2-20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2-20) had significantly enhanced neuronal affinity (IC(50) 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2-20) showed greatest Na(v)1.2 versus 1.4 selectivity (136-fold). (1)H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and an alpha-helical motif across residues 11-16 that is not found in larger mu-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller mu-conotoxin pharmacophore.     

The interactions of phenytoin and its binding site in DI-S6 segment of Na+ channel voltage-gated peptide by NMR spectroscopy and molecular modeling study.

Nuclear magnetic resonance (NMR) spectra of a model peptide (BL-DIS6), in the presence of anticonvulsant diphenyl drug, phenytoin (DPH), were measured to obtain the interactions between the selected drug and the model peptide. BL-DIS6's sequence corresponds to the S6 segment in domain I of rat brain type IIA Na+-channel. NMR studies have demonstrated that the magnitude of the chemical shifts of amide- and alpha-protons can be used as a measurement of the complex stability and binding site of the peptide. Our NMR results propose a 3(10)-helical structure for BL-DIS6, and suggest a binding cavity for DPH that involves the hydrophobic particles of residues Ans-7, Leu-8, Val-11, and Val-12. Furthermore, molecular modeling was performed to provide a possible complex conformation that the phenyl portion of DPH is accommodated in the proximity of the C-terminal residues Ala-11 and Val-12, and simultaneously the heterocyclic amine ring of DPH is perching at the residue Asn-7 periphery and stabilizing the phenyl portion deep insertion into the peptide.     

Interaction of heparin with two synthetic peptides that neutralize the anticoagulant activity of heparin

Two synthetic analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (Ac-SRGKAKVKAKVKDQTK-NH2) and the all-d-amino acid version of the same peptide (l-HIPAP and d-HIPAP, respectively) were synthesized, and their efficacy as agents for neutralization of the anticoagulant activity of heparin was assayed. The two analogue peptides were found to be equally effective for neutralization of the anticoagulant activity of heparin, as measured by restoration of the activity of serine protease factor Xa by the Coatest heparin method. The finding that l-HIPAP and d-HIPAP are equally effective suggests that d-amino acid peptides show promise as proteolytically stable therapeutic agents for neutralization of the anticoagulant activity of heparin. The interaction of l-HIPAP and d-HIPAP with heparin was characterized by 1H NMR, isothermal titration calorimetry (ITC), and heparin affinity chromatography. The two peptides were found to interact identically with heparin. Analysis of the dependence of heparin-peptide binding constants on Na+ concentration by counterion condensation theory indicates that, on average, 2.35 Na+ ions are displaced from heparin per peptide molecule bound and one peptide molecule binds per hexasaccharide segment of heparin. The analysis also indicates that both ionic and nonionic interactions contribute to the binding constant, with the ionic contribution decreasing as the Na+ concentration increases.     

Characterization of selectivity and pharmacophores of type 1 sea anemone toxins by screening seven Na(v) sodium channel isoforms

During their evolution, animals have developed a set of cysteine-rich peptides capable of binding various extracellular sites of voltage-gated sodium channels (VGSC). Sea anemone toxins that target VGSCs delay their inactivation process, but little is known about their selectivities. Here we report the investigation of three native type 1 toxins (CGTX-II, δ-AITX-Bcg1a and δ-AITX-Bcg1b) purified from the venom of Bunodosoma cangicum. Both δ-AITX-Bcg1a and δ-AITX-Bcg1b toxins were fully sequenced. The three peptides were evaluated by patch-clamp technique among Nav1.1-1.7 isoforms expressed in mammalian cell lines, and their preferential targets are Na(v)1.5>1.6>1.1. We also evaluated the role of some supposedly critical residues in the toxins which would interact with the channels, and observed that some substitutions are not critical as expected. In addition, CGTX-II and δ-AITX-Bcg1a evoke different shifts in activation/inactivation Boltzmann curves in Nav1.1 and 1.6. Moreover, our results suggest that the interaction region between toxins and VGSCs is not restricted to the supposed site 3 (S3-S4 linker of domain IV), and this may be a consequence of distinct surface of contact of each peptide vs. targeted channel. Our data suggest that the contact surfaces of each peptide may be related to their surface charges, as CGTX-II is more positive than δ-AITX-Bcg1a and δ-AITX-Bcg1b.     

Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.     

Prolylpeptide binding by the prokaryotic SH3-like domain of the diphtheria toxin repressor: a regulatory switch

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.     

Structural analysis of the heparin-binding site of the NC1 domain of collagen XIV by CD and NMR.

Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an alpha-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic alpha-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial alpha-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG.     

Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.     

Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.     

Three-dimensional solution structure of omega-conotoxin TxVII, an L-type calcium channel blocker

We determined the three-dimensional structure of omega-conotoxin TxVII, a 26-residue peptide that is an L-type calcium channel blocker, by (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 411 distance constraints obtained from nuclear Overhauser effect connectivities, 20 torsion angle constraints, and 21 constraints associated with hydrogen bonds and disulfide bonds. The root-mean-square deviations about the averaged coordinates of the backbone atoms (N, C(alpha), C, and O) and all heavy atoms were 0.50 +/- 0.09 A and 0.99 +/- 0.13 A, respectively. The structure of omega-conotoxin TxVII is composed of a triple-stranded antiparallel beta-sheet and four turns. The three disulfide bonds in omega-conotoxin TxVII form the classical cystine knot motif of toxic or inhibitory polypeptides. The overall folding of omega-conotoxin TxVII is similar to those of the N-type calcium channel blockers, omega-conotoxin GVIA and MVIIA, despite the low amino acid sequence homology among them. omega-Conotoxin TxVII exposes many hydrophobic residues to a certain surface area. In contrast, omega-conotoxin GVIA and MVIIA expose basic residues in the same way as omega-conotoxin TxVII. The channel binding site of omega-conotoxin TxVII is different from those of omega-conotoxin GVIA and MVIIA, although the overall folding of these three peptides is similar. The gathered hydrophobic residues of omega-conotoxin TxVII probably interact with the hydrophobic cluster of the alpha(1) subunit of the L-type calcium channel, which consists of 13 residues located in segments 5 and 6 in domain III and in segment 6 in domain IV.     

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.     

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Conformational Determinants of Phosphotyrosine Peptides Complexed with the Src SH2 Domain

The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA) to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the “two-pronged plug two-hole socket” model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an α-helix. In contrast, a β-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.     

Characterization of a novel weak interaction between MUC1 and Src-SH3 using nuclear magnetic resonance spectroscopy

Breast cancer causes death through cancer cell migration and subsequent metastasis to distant organs. In vitro, the MUC1 mucin can mediate breast cancer cell migration by binding to intercellular adhesion molecule-1 (ICAM-1). This migration is dependent on MUC1 cytoplasmic domain (MUC1-CD) activation of the non-receptor tyrosine kinase, Src, possibly through competitive displacement of an inhibitory Src intramolecular SH3 binding. Therefore, we characterized the binding site and affinity of the MUC1-CD for Src-SH3 using multidimensional nuclear magnetic resonance (NMR) spectroscopy to monitor the titration of the (15)N labeled Src-SH3 domain with synthetic native and mutant peptides of MUC1-CD. The results revealed that the dissociation constant (K(d)) for the interaction of the native MUC1-CD peptides and Src-SH3 domain was weak with a K(d) of 2-3 mM. Notably, the SH3 residues most perturbed upon peptide binding were located outside the usual hydrophobic binding cleft in a previously described alternate binding site on the Src-SH3, suggesting that MUC1-CD binds to a non-canonical site. The binding characteristics outlined here suggest that the interaction between Src-SH3 and MUC1-CD represents a novel weak electrostatic interaction of the type which is increasingly recognized as important in transient and dynamic protein complexes required for cell migration and signal transduction. As such, this study forms the foundation for the design of specific inhibitors of this interaction which may target breast cancer metastases with exquisite specificity.     

Crystal structure of the SH3 domain of betaPIX in complex with a high affinity peptide from PAK2

The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of betaPIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of betaPIX at 0.92 A and 1.3A resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published betaPIX/Cbl-b complex structure, and suggest the existence of a molecular switch.     

Molecular basis of isoform-specific micro-conotoxin block of cardiac,skeletal muscle,and brain Na+ channels

mu-Conotoxins (mu-CTXs) block skeletal muscle Na(+) channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na(+) channels. Although a number of conserved pore residues are recognized as critical determinants of mu-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (mu1, Na(v)1.4), human heart (hh1, Na(v)1.5), and rat brain (rb1, Na(v)1.1) Na(+) channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Na(v)1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Na(v)1.1 and Na(v)1.5. The Na(v)1.4-->Na(v)1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Na(v)1.4 numbering) reduced block of Na(v)1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Na(v)1.5--> Na(v)1.4 chimeric construct in which the Na(v)1.4 DII S5-P linker replaces the analogous segment in Na(v)1.5 showed enhanced mu-CTX block. However, these variant determinants are conserved between Na(v)1.1 and Na(v)1.4 and thus cannot explain their different sensitivities to mu-CTX. Comparison of their sequences reveals two variants at Na(v)1.4 positions 729 and 732: Ser and Asn in Na(v)1.4 compared with Thr and Lys in Na(v)1.1, respectively. The double mutation S729T/N732K rendered Na(v)1.4 more "brain-like" (30-fold downward arrow in block), and the converse mutation T925S/K928N in Na(v)1.1 reproduced the high affinity blocking phenotype of Na(v)1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in mu-CTX binding, thus shaping isoform-specific toxin sensitivity.