RESUMO
κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey's excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4-5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.
Assuntos
Conotoxinas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Conotoxinas/química , Oócitos , Concentração Osmolar , Bloqueadores dos Canais de Potássio/farmacologia , Ligação Proteica , Superfamília Shaker de Canais de Potássio/química , Xenopus laevisRESUMO
Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.
Assuntos
Venenos de Moluscos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Caramujo Conus , Ativação do Canal Iônico , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/genética , Canal de Potássio Kv1.4/metabolismo , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Canal de Potássio Kv1.6/antagonistas & inibidores , Canal de Potássio Kv1.6/genética , Canal de Potássio Kv1.6/metabolismo , Potenciais da Membrana , Oócitos , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.
Assuntos
Caramujo Conus/química , Venenos de Moluscos/química , Neuropeptídeos/farmacologia , Peptídeos/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Baculoviridae , Células HEK293 , Humanos , Venenos de Moluscos/síntese química , Venenos de Moluscos/farmacologia , Neuropeptídeos/síntese química , Neuropeptídeos/química , Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Células Sf9/virologiaRESUMO
In the last years it has been increasingly clear that KV-channel activity modulates neurotransmitter release. The subcellular localization and composition of potassium channels are crucial to understanding its influence on neurotransmitter release. To investigate the role of KV in corticostriatal synapses modulation, we combined extracellular recording of population-spike and pharmacological blockage with specific and nonspecific blockers to identify several families of KV channels. We induced paired-pulse facilitation (PPF) and studied the changes in paired-pulse ratio (PPR) before and after the addition of specific KV blockers to determine whether particular KV subtypes were located pre- or postsynaptically. Initially, the presence of KV channels was tested by exposing brain slices to tetraethylammonium or 4-aminopyridine; in both cases we observed a decrease in PPR that was dose dependent. Further experiments with tityustoxin, margatoxin, hongotoxin, agitoxin, dendrotoxin, and BDS-I toxins all rendered a reduction in PPR. In contrast heteropodatoxin and phrixotoxin had no effect. Our results reveal that corticostriatal presynaptic KV channels have a complex stoichiometry, including heterologous combinations KV1.1, KV1.2, KV1.3, and KV1.6 isoforms, as well as KV3.4, but not KV4 channels. The variety of KV channels offers a wide spectrum of possibilities to regulate neurotransmitter release, providing fine-tuning mechanisms to modulate synaptic strength.
Assuntos
Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Terminações Pré-Sinápticas/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Canais de Potássio Shaw/fisiologia , Sinapses/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Masculino , Bloqueadores dos Canais de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Ratos , Ratos Wistar , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Canais de Potássio Shaw/antagonistas & inibidores , Sinapses/efeitos dos fármacosRESUMO
Several potassium (K(+)) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K(+) channels allow sodium reabsorption in the proximal tubule (PT), K(+) recycling and K(+) reabsorption in the thick ascending limb (TAL) and K(+) secretion and K(+) reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K(+) to function as a secretory K(+) channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K(+) channels. Our results expand the repertoire of K(+) channels that contribute to K(+) homeostasis to include the Kv1 family.
Assuntos
Perfilação da Expressão Gênica , Família Multigênica , Néfrons/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Animais , Células Endoteliais/metabolismo , Imunofluorescência , Immunoblotting , Córtex Renal/metabolismo , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Masculino , Microscopia Confocal , Podócitos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superfamília Shaker de Canais de Potássio/metabolismoRESUMO
TASK channels belong to the family of K(+) channels with 4 transmembrane segments and 2 pore domains (4TM/2P) per subunit. These channels have been related to apoptosis in cerebellar granule neurons (CGN), as well as cancer in other tissues. TASK current is regulated by hormones, neurotransmitters, anesthetics and divalent cations, which are not selective. Recently, there has been found some organic compounds that inhibit TASK current selectively. In order to find other modulators, we report here a group of five dihydropyrrolo[2,1-a]isoquinolines (DPIs), four of them with putative anticancer activity, that were evaluated on TASK-1 and TASK-3 channels. The compounds 1, 2 and 3 showed IC50 < 320 µM on TASK-1 and TASK-3, intermediate activity on TASK-1/TASK-3 heterodimer, moderate effect over hslo and TREK-1 (500 µM), and practically not inhibition on Shaker-IR, herg and IRK2.1 potassium channels, when they were expressed heterologously in Xenopus laevis oocytes. In rat CGN, 500 µM of these three compounds induced a decrement by >39% of the TASK-carried leak current. Finally, only compound 1 showed significant protection (â¼36%) against apoptotic death of CGN induced by K(+) deprivation. These results suggest that DPI compounds could be potential candidates for designing new selective inhibitors of TASK channels.
Assuntos
Isoquinolinas/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Pirróis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Isoquinolinas/química , Camundongos , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Pirróis/química , Ratos , Ratos Wistar , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and Kâº-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B Kâº-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of â¼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).
Assuntos
Canal de Potássio Kv1.3/antagonistas & inibidores , Peptídeos/metabolismo , Venenos de Escorpião/metabolismo , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/química , Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/metabolismo , Ligação Proteica , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificação , Escorpiões/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Potassium channels exhibit a large diversity of single-channel conductances. Shaker is a low-conductance K-channel in which Pro475âAsp, a single-point mutation near the internal pore entrance, promotes 6- to 8-fold higher unitary current. To assess the mechanism for this higher conductance, we measured Shaker-P475D single-channel current in a wide range of symmetrical K(+) concentrations and voltages. Below 300 mM K(+), the current-to-voltage relations (i-V) showed inward rectification that disappeared at 1000 mM K(+). Single-channel conductance reached a maximum of â¼190 pS at saturating [K(+)], a value 4- to 5-fold larger than that estimated for the native channel. Intracellular Mg(2+) blocked this variant with â¼100-fold higher affinity. Near zero voltage, blockade was competitively antagonized by K(+); however, at voltages >100 mV, it was enhanced by K(+). This result is consistent with a lock-in effect in a single-file diffusion regime of Mg(2+) and K(+) along the pore. Molecular-dynamics simulations revealed higher K(+) density in the pore, especially near the Asp-475 side chains, as in the high-conductance MthK bacterial channel. The molecular dynamics also showed that K(+) ions bound distally can coexist with other K(+) or Mg(2+) in the cavity, supporting a lock-in mechanism. The maximal K(+) transport rate and higher occupancy could be due to a decrease in the electrostatic energy profile for K(+) throughout the pore, reducing the energy wells and barriers differentially by â¼0.7 and â¼2 kT, respectively.
Assuntos
Condutividade Elétrica , Magnésio/farmacologia , Mutação Puntual , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Simulação de Dinâmica Molecular , Porosidade , Conformação Proteica , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/genética , Eletricidade Estática , Xenopus laevisRESUMO
To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here, we show that delayed rectifier K(+) channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very similar behavior when expressed in Xenopus oocytes. However, the transcribed messenger RNAs are extensively edited, creating functional diversity. One editing site, which recodes an isoleucine to a valine in the channel's pore, greatly accelerates gating kinetics by destabilizing the open state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus adenosine-to-inosine RNA editing can respond to the physical environment.
Assuntos
Aclimatação/genética , Octopodiformes/fisiologia , Edição de RNA , Superfamília Shaker de Canais de Potássio/fisiologia , Adenosina/metabolismo , Animais , Regiões Antárticas , Inosina/metabolismo , Dados de Sequência Molecular , Octopodiformes/genética , Proteínas Recombinantes , Superfamília Shaker de Canais de Potássio/genética , Especificidade da Espécie , Xenopus laevisRESUMO
In the nervous system, AâI RNA editing has an important role in regulating neuronal excitability. Ligand-gated membrane receptors, synaptic proteins, as well as ion channels, are targets for recoding by RNA editing. Although scores of editing sites have been identified in the mammalian brain, little is known about the functional alterations that they cause, and even less about the mechanistic underpinnings of how they change protein function. We have previously shown that an RNA editing event (I,400 V) alters the inner permeation pathway of human K(V)1.1, modifying the kinetics of fast inactivation. Here we show that the channel's inactivation gate enters deep into the ion permeation pathway and the very tip establishes a direct hydrophobic interaction with the edited position. By converting I to V, the intimacy of the interaction is reduced, allowing the inactivation gate to unbind with much faster kinetics.