RESUMEN
We have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza A and B virus and respiratory syncytial virus (RSV) via targeting of the host dihydrofolate reductase (DHFR) enzyme. Most analogues proved active against influenza B virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (EC50 = 0.060 µM), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. Activity was also observed for two influenza A strains, including a virus with the S31N mutant form of M2 proton channel, which is the most prevalent resistance mutation for amantadine. Importantly, the compounds displayed nanomolar activity against RSV and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (EC50 â¼0.008 µM), far surpassing the potency and safety profile of the licensed drug ribavirin (EC50 = 5.8 µM, SI > 43).
Asunto(s)
Antivirales/farmacología , Antagonistas del Ácido Fólico/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Proguanil/farmacología , Virus Sincitiales Respiratorios/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismo , Triazinas/farmacología , Antivirales/síntesis química , Antivirales/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/química , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Proguanil/síntesis química , Proguanil/química , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/químicaRESUMEN
New insights on the amantadine resistance mechanism of the V27A mutant were obtained through the study of novel, easily accessible 4-(1- and 2-adamantyl)piperidines, identified as dual binders of the wild-type and V27A mutant M2 channels of influenza A virus. Their antiviral activity and channel blocking ability were determined using cell-based assays and two-electrode voltage clamp (TEVC) technique on M2 channels, respectively. In addition, electrophysiology experiments revealed two interesting findings: (i) these inhibitors display a different behavior against the wild-type versus V27A mutant A/M2 channels, and (ii) the compounds display antiviral activity when they have kd equal or smaller than 10-6 while they do not exhibit antiviral activity when kd is 10-5 or higher although they may show blocking activity in the TEV assay. Thus, caution must be taken when predicting antiviral activity based on percent channel blockage in electrophysiological assays. These findings provide experimental evidence of the resistance mechanism of the V27A mutation to wild-type inhibitors, previously predicted in silico, offer an explanation for the lack of antiviral activity of compounds active in the TEV assay, and may help design new and more effective drugs.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Mutación , Animales , Perros , Farmacorresistencia Viral , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Pruebas de Sensibilidad Microbiana , Técnicas de Placa-ClampRESUMEN
The miniature channel, Kcv, is a structural equivalent of the pore of all K+ channels. Here, we follow up on a previous observation that a largely voltage-insensitive channel can be converted into a slow activating inward rectifier after extending the outer transmembrane domain by one Ala. This gain of rectification can be rationalized by dynamic salt bridges at the cytosolic entrance to the channel; opening is favored by voltage-sensitive formation of salt bridges and counteracted by their disruption. Such latent voltage sensitivity in the pore could be relevant for the understanding of voltage gating in complex Kv channels.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Cinética , Modelos Biológicos , Proteínas Mutantes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , TemperaturaRESUMEN
The M2 proton channel of influenza A virus is an integral membrane protein involved in the acidification of the viral interior, a step necessary for the release of the viral genetic material and replication of new virions. The aim of this study is to explore the mechanism of drug (un)binding to the M2 channel in order to gain insight into the structural and energetic features relevant for the development of novel inhibitors. To this end, we have investigated the binding of amantadine (Amt) to the wild type (wt) M2 channel and its V27A variant using multiple independent molecular dynamics simulations, exploratory conventional metadynamics, and multiple-walkers well-tempered metadynamics calculations. The results allow us to propose a sequential mechanism for the (un)binding of Amt to the wt M2 channel, which involves the adoption of a transiently populated intermediate (up state) leading to the thermodynamically favored down binding mode in the channel pore. Furthermore, they suggest that chloride anions play a relevant role in stabilizing the down binding mode of Amt to the wt channel, giving rise to a kinetic trapping that explains the experimentally observed pseudoirreversible inhibition of the wt channel by Amt. We propose that this trapping mechanism underlies the inhibitory activity of potent M2 channel blockers, as supported by the experimental confirmation of the irreversible binding of a pyrrolidine analogue from electrophysiological current assays. Finally, the results reveal that the thermodynamics and kinetics of Amt (un)binding is very sensitive to the V27A mutation, providing a quantitative rationale to the drastic decrease in inhibitory potency against the V27A variant. Overall, these findings pave the way to explore the inhibitory activity of Amt-related analogues in mutated M2 channel variants, providing guidelines for the design of novel inhibitors against resistant virus strains.
RESUMEN
Basic bulky amines such as amantadine are well-characterized M2 channel blockers, useful for treating influenza. Herein we report our surprising findings that charge-neutral, bulky isocyanides exhibit activities similar to--or even higher than--that of amantadine. We also demonstrate that these isocyanides have potent growth inhibitory activity against the H5N1 virus. The -NH2 to -N≡C group replacement within current anti-influenza drugs was found to give compounds with high activities at low-micromolar concentrations. For example, a tenfold improvement in potency was observed for 1-isocyanoadamantane (27), with an EC50 value of 0.487â µm against amantadine-sensitive H5N1 virus as determined by both MTT and plaque-reduction assays, without showing cytotoxicity. Furthermore, the isocyanide analogues synthesized in this study did not inhibit the V27A or S31N mutant M2 ion channels, according to electrophysiology experiments, and did not exhibit activity against amantadine-resistant virus strains.
Asunto(s)
Antivirales/farmacología , Cianuros/farmacología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Proteínas de la Matriz Viral/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Cianuros/síntesis química , Cianuros/química , Perros , Relación Dosis-Respuesta a Droga , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby/microbiología , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Proteínas de la Matriz Viral/metabolismoRESUMEN
The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.
Asunto(s)
Optogenética , Proteínas Recombinantes de Fusión/efectos de la radiación , Animales , Avena/metabolismo , Fenómenos Biofísicos , Células HEK293 , Humanos , Larva , Luz , Fototropinas/química , Fototropinas/genética , Canales de Potasio/química , Canales de Potasio/genética , Conformación Proteica/efectos de la radiación , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Virales/química , Proteínas Virales/genética , Pez CebraRESUMEN
Two new polycyclic scaffolds were synthesized and evaluated as anti-influenza A compounds. The 5-azapentacyclo[6.4.0.0(2,10).0(3,7).0(9,11)]dodecane derivatives were only active against the wild-type M2 channel in the low-micromolar range. However, some of the 14-azaheptacyclo[8.6.1.0(2,5).0(3,11).0(4,9).0(6,17).0(12,16)]heptadecane derivatives were dual inhibitors of the wild-type and the V27A mutant M2 channels. The antiviral activity of these molecules was confirmed by cell culture assays. Their binding mode was analysed through molecular dynamics simulations, which showed the existence of distinct binding modes in the wild type M2 channel and its V27A variant.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Compuestos Policíclicos/farmacología , Proteínas de la Matriz Viral/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Sitios de Unión/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Perros , Relación Dosis-Respuesta a Droga , Virus de la Influenza A/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mutación , Compuestos Policíclicos/síntesis química , Compuestos Policíclicos/química , Relación Estructura-Actividad , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
Signals recorded at the cell membrane are meaningful indicators of the physiological vs. pathological state of a cell and will become useful diagnostic elements in nanomedicine. In this project we present a coherent strategy for the design and fabrication of a bio-nano-sensor that monitors changes in intracellular cell calcium concentration and allows an easy read out by converting the calcium signal into an electrical current in the range of microampere that can be easily measured by conventional cell electrophysiology apparatus.
Asunto(s)
Técnicas Biosensibles , Calcio/aislamiento & purificación , Canales de Potasio/química , Calcio/química , Señalización del Calcio/fisiología , Potenciales de la Membrana , Canales de Potasio/fisiologíaRESUMEN
Two alternative syntheses of 2-oxaadamantan-5-amine, a novel analog of the clinically approved drug amantadine, are reported. The compound has been tested as an anti-influenza A virus agent and as an NMDA receptor antagonist. While the compound was not antivirally active, it displayed moderate activity as an NMDA receptor antagonist.
RESUMEN
The synthesis of several [4,4,3], [4,3,3], and [3,3,3]azapropellanes is reported. Several of the novel amines displayed low-micromolar activities against an amantadine-resistant H1N1 strain, but they did not show activity against an amantadine-sensitive H3N2 strain. None of the tested compounds inhibit the influenza A/M2 proton channel function. Most of the compounds did not show cytotoxicity for MDCK cells.
RESUMEN
The current of the minimal viral K(+) channel Kcv(PCBV-1) heterologously expressed in Xenopus oocytes is strongly inhibited by reactive oxygen species (ROS) like H(2)O(2). Possible targets for the ROS effect are two cysteines (C53 and C79) and four methionines (M1, M15, M23, and M26). The C53A/C79A and M23L/M26L double mutations maintained the same ROS sensitivity as the wild type. However, M15L as a single mutant or in combination with C53A/C79A and/or M23L/M26L caused a complete loss of sensitivity to H(2)O(2). These results indicate a prominent role of M15 at the cytosolic end of the outer transmembrane helix for gating of Kcv(PCBV-1). Furthermore, even though the channel lacks a canonical voltage sensor, it exhibits a weak voltage sensitivity, resulting in a slight activation in the millisecond range after a voltage step to negative potentials. The M15L mutation inverts this kinetics into an inactivation, underlining the critical role of this residue for gating. The negative slope of the I-V curves of M15L is the same as in the wild type, indicating that the selectivity filter is not involved.
Asunto(s)
Canales de Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Peróxido de Hidrógeno/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Canales de Potasio/química , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/químicaRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2-4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V(½) in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN channels.
Asunto(s)
AMP Cíclico/metabolismo , Activación del Canal Iónico/fisiología , Canales Iónicos/química , Canales Iónicos/metabolismo , Multimerización de Proteína/fisiología , Animales , AMP Cíclico/química , AMP Cíclico/genética , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/genética , Oocitos , Canales de Potasio , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Xenopus laevisRESUMEN
BACKGROUND: Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. METHODOLOGY/PRINCIPAL FINDINGS: We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T-->S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. CONCLUSIONS/SIGNIFICANCE: The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features.
Asunto(s)
Amantadina/farmacología , Bario/farmacología , Canales de Potasio/química , Proteínas Virales/fisiología , Animales , Sitios de Unión , Electrofisiología/métodos , Modelos Genéticos , Mutación , Oocitos/metabolismo , Pichia/metabolismo , Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/fisiología , Proteínas Virales/química , Proteínas Virales/metabolismo , Xenopus laevis/metabolismoRESUMEN
Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114+/-11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. "Fast" gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel current-voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a "slow" gating is revealed by the very low (in the order of 1-4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio/metabolismo , Proteínas Virales/metabolismo , Animales , Oocitos , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Chlorella virus PBCV-1 (Paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In this paper, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisiae mutants that lack endogenous K+ uptake systems. Compared with the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, barium and caesium. Single channel analyses reveal a stochastic gating behaviour and a voltage-dependent conductance that resembles the macroscopic I/V relationship. One difference between PBCV-1 and ATCV-1 Kcv is that the latter is more permeable to K+ than Rb+. This difference is partially explained by the presence of a tyrosine residue in the selective filter of ATCV-1 Kcv, whereas PBCV-1 Kcv has a phenylalanine. Hence, ATCV-1 Kcv is the smallest protein to form a K+ channel and it will serve as a model for studying structure-function correlations inside the potassium channel pore.
Asunto(s)
Chlorella/virología , Canales de Potasio/fisiología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Bario/farmacología , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/fisiología , Femenino , Prueba de Complementación Genética , Transporte Iónico , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/química , Canales de Potasio/genética , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Homología de Secuencia de Aminoácido , Proteínas Virales/química , Proteínas Virales/genética , Xenopus laevisRESUMEN
14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Animales , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Cesio/metabolismo , Electrofisiología/métodos , Escherichia coli/metabolismo , Activación del Canal Iónico , Mutación , Oocitos/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Recombinantes/química , Xenopus , Quinasas raf/químicaRESUMEN
An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.
Asunto(s)
Acetabularia/fisiología , Potenciales de la Membrana/fisiología , Bombas de Protones/fisiología , Rodopsina/fisiología , Acetabularia/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Luz , Biología Marina , Potenciales de la Membrana/efectos de la radiación , Bombas de Protones/efectos de la radiación , Dosis de Radiación , Rodopsina/efectos de la radiación , Agua de Mar/microbiologíaRESUMEN
Fast and selective transport of water through cell membranes is facilitated by water channels. Water channels belonging to the major intrinsic proteins (MIPs) family have been found in all three domains of life, Archaea, Bacteria, and Eukarya. Here we show that Chlorella virus MT325 has a water channel gene, aqpv1, that forms a functional aquaglyceroporin in oocytes. aqpv1 is transcribed during infection together with MT325 kcv, a gene encoding a previously undescribed type of viral potassium channel. Coexpression of AQPV1 and MT325-Kcv in Xenopus oocytes synergistically increases water transport, suggesting a possible concerted action of the two channels in the infection cycle. The two channels operate by a thermodynamically coupled mechanism that simultaneously alters water conductance and driving force for water movement. Considering the universal role of osmosis, this mechanism is relevant to any cell coexpressing water and potassium channels and could have pathological as well as basic physiological relevance.
Asunto(s)
Acuagliceroporinas/genética , Virus ADN/genética , Canales de Potasio/genética , Agua/metabolismo , Secuencia de Aminoácidos , Animales , Acuagliceroporinas/química , Virus ADN/metabolismo , Genes Virales , Datos de Secuencia Molecular , Ósmosis , Canales de Potasio/química , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.
Asunto(s)
Membrana Celular/fisiología , Chlorella/fisiología , Chlorella/virología , Phycodnaviridae/fisiología , Canales de Potasio/fisiología , ADN Viral/metabolismo , Cinética , Potenciales de la Membrana , Bloqueadores de los Canales de Potasio/farmacologíaRESUMEN
Kcv, isolated from a Chlorella virus, is the smallest known K+ channel. When Kcv is expressed in Xenopus oocytes and exposed to 50 mM: [K+](o), its open-state current-voltage relationship (I-V) has the shape of a "tilted S" between -200 and +120 mV. Details of this shape depend on the conditioning voltage (V (c)) immediately before an I-V recording. Unexpectedly, the I-V relationships, recorded in different [K+](o), do intersect. These characteristics are numerically described here by fits of a kinetic model to the experimental data. In this model, the V (c) sensitivity of I-V is mainly assigned to an affinity increase of external K+ association at more positive voltages. The general, tilted-S shape as well as the unexpected intersections of the I-V relationships are kinetically described by a decrease of the cord conductance by the electrochemical driving force for K+ in either direction, like in fast V-dependent blocking by competing ions.