Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na_Vs, are responsible for the rapid rise of action potentials in excitable tissues. Na_V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na_V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na_V1.4 (skeletal muscle) and Na_V1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na_V1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na_V isoform has been challenging because they usually show undesired crossreactivity for different Na_V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa_V1.4 or CTNa_V1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa_V1.4 and CTNa_V1.5 with high affinity (K_D ∼ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na_V1.4 and Na_V1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na_V1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na_V reagents to capture Na_Vs from cell lysates and as molecular visualization agents for Na_Vs.     

Molecular dynamics simulations on the interaction of the transmembrane NavAb channel with cholesterol and lipids in the membrane

Increased cholesterol levels are associated with multiple pathological conditions. In this work, molecular dynamics simulations were applied to observe the influence of membrane cholesterol levels on a voltage-gated sodium channel. Different lipid compositions are modeled around the channel to obtain information about the possible effects by which cholesterol influences Na_vAb channels. Cholesterol was normally not directly interacting with either the closed or inactivated conformation. Cholesterol increased lipid packing implying that it plays a crucial role in restricting lipid movement in the region around 1 nm of the channel in a 1-palmitoyl-2-oeleoyl phosphatidylcholine matrix. Our results provide the first computational indication of an indirect modulation of Na_vAb channels by membrane cholesterol.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Intercellular Electrical Communication in the Heart: A New,Active Role for the Intercalated Disk

Abstract

Cardiac conduction is the propagation of electrical excitation through the heart and is responsible for triggering individual myocytes to contract in synchrony. Canonically, this process has been thought to occur electrotonically, by means of direct flow of ions from cell to cell. The intercalated disk (ID), the site of contact between adjacent myocytes, has been viewed as a structure composed of mechanical junctions that stabilize the apposition of cell membranes and gap junctions which constitute low resistance pathways between cells. However, emerging evidence suggests a more active role for structures within the ID in mediating intercellular electrical communication by means of non-canonical ephaptic mechanisms. This review will discuss the role of the ID in the context of the canonical, electrotonic view of conduction and highlight new, emerging possibilities of its playing a more active role in ephaptic coupling between cardiac myocytes.     

X-ray structure and mutagenesis of the scorpion depressant toxin LqhIT2 reveals key determinants crucial for activity and anti-insect selectivity

Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.     

Determination of potential selective inhibitors for ROCKI and ROCKII isoforms with molecular modeling techniques: structure based docking,ADMET and molecular dynamics simulation

Rho-associated protein kinases (ROCKs) are a member of the serine/threonine protein kinase family and potential therapeutic target for various diseases. This enzyme has two isoforms, Rho-associated protein kinase I (ROCKI) and Rho-associated protein kinase II (ROCKII). They share an overall 65% homology in all amino acid sequence and 92% homology in kinase domains. Since, the kinase domains of ROCKI and ROCKII are highly conserved and similar, the discovery and design of isoform-selective inhibitors are more challenging. Thus, most currently available agents that is against ROCKs exhibit low selectivity and severe side effects. Therefore, this study aimed to elucidate the interaction of compounds that indicated high potential in experimental studies against ROCKI and ROCKII enzymes in the molecular level with molecular modeling techniques. Firstly, we determined the interaction property of catalytic sites of the ROCKs by analyzing with molecular docking. Based on these results, the best ligands (50 compounds) corresponding to experimental studies were selected, and then absorption, distribution, metabolism and excretion – toxicity (ADMET) analysis of these compounds were implemented. According to these study results, the compound 40 for ROCKI and the compound 50 for ROCKII were identified as selective and highly potent inhibitors. And finally, molecular dynamics (MD) simulations were performed for the stability of ROCKs with identified compounds. In the light of this study, it will be possible to treat diseases that ROCKs have a role by developing more effective and specific ROCK inhibitors.

Communicated by Ramaswamy H. Sarma     

Strategy and validation of a structure-based method for the discovery of selective inhibitors of PAK isoforms and the evaluation of their anti-cancer activity

p21 activated kinase 4 (PAK4), which belongs to the serine/threonine (Ser/Thr) protein kinase family, is a representative member of the PAK family and plays a significant role in multiple processes associated with cancer development. In this study, structure-based virtual screening was performed to discover novel and selective small molecule scaffolds, and a 6-hydroxy-2-mercapto-3-phenylpyrimidin-4(3H)-one-based compound (SPU-106, 14#) was identified as an effective PAK4 inhibitor. By combining both a molecular docking study and molecular dynamics (MD) simulation strategies, the binding mode was determined in the PAK4 site. The SPU-106 compound could efficiently and selectively bind to the PAK4 kinase domain at an IC₅₀ of 21.36 μM according to the kinase analysis. The designed molecular probe demonstrated that SPU-106 binds to the kinase domain in the C-terminus of PAK4. Further investigation revealed that the SPU-106 had a strong inhibitory effect on the invasion of SGC7901 cells but without any cytotoxicity. The western blot analysis indicated that the compound potently inhibited the PAK4/LIMK1/cofilin and PAK4/SCG10 signaling pathways. Thus, our work shows the successful application of computational strategies for the discovery of selective hits, and SPU-106 may be an effective PAK4 inhibitor for further development as an antitumor agent.     

Histidine residues in the IS3-IS4 loop are critical for nickel-sensitive inhibition of the Cav2.3 calcium channel

We recently reported that a histidine (H191) in the S3-S4 loop of domain I is critical for nickel inhibition of the Cav3.2 T-type Ca2+ channel. As in Cav3.2, two histidine residues are commonly found in the IS3-IS4 loops of mammalian Cav2.3 Ca2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Cav2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3-IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Cav2.3.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).