RESUMEN
Kv1.3 K+ channels play a central role in the regulation of T cell activation and Ca2+ signaling under physiological and pathophysiological conditions. Peptide toxins targeting Kv1.3 have a significant therapeutic potential in the treatment of autoimmune diseases; thus, the discovery of new toxins is highly motivated. Based on the transcriptome analysis of the venom gland of V. mexicanus smithi a novel synthetic peptide, sVmKTx was generated, containing 36 amino acid residues. sVmKTx shows high sequence similarity to Vm24, a previously characterized peptide from the same species, but contains a Glu at position 32 as opposed to Lys32 in Vm24. Vm24 inhibits Kv1.3 with high affinity (Kd = 2.9 pM). However, it has limited selectivity (~1,500-fold) for Kv1.3 over hKv1.2, hKCa3.1, and mKv1.1. sVmKTx displays reduced Kv1.3 affinity (Kd = 770 pM) but increased selectivity for Kv1.3 over hKv1.2 (~9,000-fold) as compared to Vm24, other channels tested in the panel (hKCa3.1, hKv1.1, hKv1.4, hKv1.5, rKv2.1, hKv11.1, hKCa1.1, hNav1.5) were practically insensitive to the toxin at 2.5 µM. Molecular dynamics simulations showed that introduction of a Glu instead of Lys at position 32 led to a decreased structural fluctuation of the N-terminal segment of sVmKTx, which may explain its increased selectivity for Kv1.3. sVmKTx at 100 nM concentration decreased the expression level of the Ca2+ -dependent T cell activation marker, CD40 ligand. The high affinity block of Kv1.3 and increased selectivity over the natural peptide makes sVmKTx a potential candidate for Kv1.3 blockade-mediated treatment of autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes , Venenos de Escorpión , Perfilación de la Expresión Génica , Humanos , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Linfocitos T/metabolismoRESUMEN
Mutation-induced transformations of conductivity and selectivity in NaChBac bacterial channels are studied experimentally and interpreted within the framework of ionic Coulomb blockade (ICB), while also taking account of resonant quantised dehydration (QD) and site protonation. Site-directed mutagenesis and whole-cell patch-clamp experiments are used to investigate how the fixed charge Qf at the selectivity filter (SF) affects both valence selectivity and same-charge selectivity. The new ICB/QD model predicts that increasing â£Qf⣠should lead to a shift in selectivity sequences toward larger ion sizes, in agreement with the present experiments and with earlier work. Comparison of the model with experimental data leads to the introduction of an effective charge Qf∗ at the SF, which was found to differ between Aspartate and Glutamate charged rings, and also to depend on position within the SF. It is suggested that protonation of the residues within the restricted space of the SF is important in significantly reducing the effective charge of the EEEE ring. Values of Qf∗ derived from experiments on divalent blockade agree well with expectations based on the ICB/QD model and have led to the first demonstration of ICB oscillations in Ca2+ conduction as a function of the fixed charge. Preliminary studies of the dependence of Ca2+ conduction on pH are qualitatively consistent with the predictions of the model.
Asunto(s)
Secuencia de Aminoácidos/genética , Líquidos Iónicos/química , Canales de Sodio/química , Ácido Aspártico/química , Calcio/metabolismo , Ácido Glutámico/química , Iones/química , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Canales de Sodio/genéticaRESUMEN
TPC2-A1-N and TPC2-A1-P, two novel small molecules, differentially activate two-pore channel 2 (TPC2) and mimic the activation of TPC2 with NAADP and PIP2, resulting in distinct ion channel selectivities. These two different modes of TPC2 activity have physiological, and possibly pathophysiological, implications as they can modulate vesicle trafficking and lysosomal exocytosis.
Asunto(s)
Canales de Calcio/metabolismo , Animales , Agonistas de los Canales de Calcio/química , Agonistas de los Canales de Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Humanos , Iones , Modelos MolecularesRESUMEN
A key driving force for ion channel selectivity is represented by the negative charge of the Selectivity Filter carried by aspartate (D) and glutamate (E) residues. However, the structural effects and specific properties of D and E residues have not been extensively studied. In order to investigate this issue we studied the mutants of NaChBac channel with all possible combinations of D and E in the charged rings in position 191 and 192. Electrophysiological measurements showed significant Ca2+ currents only when position 191 was occupied by E. Equilibrium Molecular Dynamics simulations revealed the existence of two binding sites, corresponding to the charged rings and another one, more internal, at the level of L190. The simulations showed that the ion in the innermost site can interact with the residue in position 191 only when this is glutamate. Based on the MD simulations, we suggest that a D in position 191 leads to a high affinity Ca2+ block site resulting from a significant drop in the free energy of binding for an ion moving between the binding sites; in contrast, the free energy change is more gradual when an E residue occupies position 191, resulting in Ca2+ permeability. This scenario is consistent with the model of ion channel selectivity through stepwise changes in binding affinity proposed by Dang and McCleskey. Our study also highlights the importance of the structure of the selectivity filter which should contribute to the development of more detailed physical models for ion channel selectivity.
Asunto(s)
Ácido Aspártico/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Permeabilidad de la Membrana Celular , Ácido Glutámico/metabolismo , Canales de Sodio/química , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/metabolismo , Cationes , Cricetinae , Cricetulus , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Sodio/metabolismo , Relación Estructura-Actividad , Factores de Tiempo , Agua/químicaRESUMEN
The Kv1.3 channel-acting scorpion toxins usually adopt the conserved anti-parallel ß-sheet domain as the binding interface, but it remains challenging to discover some highly selective Kv1.3 channel-acting toxins. In this work, we investigated the pharmacological profile of the Kv1.3 channel-acting BmKTX-D33H, a structural analogue of the BmKTX scorpion toxin. Interestingly, BmKTX-D33H, with its conserved anti-parallel ß-sheet domain as a Kv1.3 channel-interacting interface, exhibited more than 1000-fold selectivity towards the Kv1.3 channel as compared to other K⺠channels (including Kv1.1, Kv1.2, Kv1.7, Kv11.1, KCa2.2, KCa2.3, and KCa3.1). As expected, BmKTX-D33H was found to inhibit the cytokine production and proliferation of both Jurkat cells and human T cells in vitro. It also significantly improved the delayed-type hypersensitivity (DTH) responses, an autoreactive T cell-mediated inflammation in rats. Amino acid sequence alignment and structural analysis strongly suggest that the "evolutionary" Gly11 residue of BmKTX-D33H interacts with the turret domain of Kv1 channels; it appears to be a pivotal amino acid residue with regard to the selectivity of BmKTX-D33H towards the Kv1.3 channel (in comparison with the highly homologous scorpion toxins). Together, our data indicate that BmKTX-D33H is a Kv1.3 channel-specific blocker. Finally, the remarkable selectivity of BmKTX-D33H highlights the great potential of evolutionary-guided peptide drug design in future studies.
Asunto(s)
Factores Inmunológicos/farmacología , Canal de Potasio Kv1.3/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes , Complejo CD3 , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Femenino , Células HEK293 , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/tratamiento farmacológico , Factores Inmunológicos/química , Factores Inmunológicos/uso terapéutico , Células Jurkat , Ovalbúmina , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/uso terapéutico , Ratas Endogámicas Lew , Venenos de Escorpión/química , Venenos de Escorpión/uso terapéutico , Escorpiones , Alineación de Secuencia , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
A putative porin function has been assigned to VCA1008 of Vibrio cholerae. Its coding gene, vca1008, is expressed upon colonization of the small intestine in infant mice and human volunteers, and is essential for infection. In vitro, vca1008 is expressed under inorganic phosphate limitation and, in this condition, VCA1008 is the major outer membrane protein of the bacterium. Here, we provide the first functional characterization of VCA1008 reconstituted into planar lipid bilayers. Our main findings were: 1) VCA1008 forms an ion channel that, at high voltage (~±100 mV), presents a voltage-dependent activity and displays closures typical of trimeric porins, with a conductance of 4.28±0.04 nS (n=164) in 1M KCl; 2) It has a preferred selectivity for anions over cations; 3) Its conductance saturates with increasing inorganic phosphate concentration, suggesting VCA1008 contains binding site(s) for this anion; 4) Its ion selectivity is controlled by both fixed charged residues within the channel and diffusion along the pore; 5) Partitioning of poly (ethylene glycol)s (PEGs) of different molecular mass suggests that VCA1008 channel has a pore exclusion limit of 0.9 nm.