RESUMEN
In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio , Arginina/metabolismo , Activación del Canal Iónico/genética , Simulación de Dinámica Molecular , MutaciónRESUMEN
Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population with high immunosuppressive activity that proliferates in infections, inflammation, and tumor microenvironments. In tumors, MDSC exert immunosuppression mainly by producing reactive oxygen species (ROS), a process triggered by the NADPH oxidase 2 (NOX2) activity. NOX2 is functionally coupled with the Hv1 proton channel in certain immune cells to support sustained free-radical production. However, a functional expression of the Hv1 channel in MDSC has not yet been reported. Here, we demonstrate that mouse MDSC express functional Hv1 proton channel by immunofluorescence microscopy, flow cytometry, and Western blot, besides performing a biophysical characterization of its macroscopic currents via patch-clamp technique. Our results show that the immunosuppression by MDSC is conditional to their ability to decrease the proton concentration elevated by the NOX2 activity, rendering Hv1 a potential drug target for cancer treatment.
Asunto(s)
Canales Iónicos , Células Supresoras de Origen Mieloide , Protones , Linfocitos T , Animales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ratones , Células Supresoras de Origen Mieloide/inmunología , NADPH Oxidasa 2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunologíaRESUMEN
Large-conductance Ca2+- and voltage-activated K+ (BK) channels play many physiological roles ranging from the maintenance of smooth muscle tone to hearing and neurosecretion. BK channels are tetramers in which the pore-forming α subunit is coded by a single gene (Slowpoke, KCNMA1). In this review, we first highlight the physiological importance of this ubiquitous channel, emphasizing the role that BK channels play in different channelopathies. We next discuss the modular nature of BK channel-forming protein, in which the different modules (the voltage sensor and the Ca2+ binding sites) communicate with the pore gates allosterically. In this regard, we review in detail the allosteric models proposed to explain channel activation and how the models are related to channel structure. Considering their extremely large conductance and unique selectivity to K+, we also offer an account of how these two apparently paradoxical characteristics can be understood consistently in unison, and what we have learned about the conduction system and the activation gates using ions, blockers, and toxins. Attention is paid here to the molecular nature of the voltage sensor and the Ca2+ binding sites that are located in a gating ring of known crystal structure and constituted by four COOH termini. Despite the fact that BK channels are coded by a single gene, diversity is obtained by means of alternative splicing and modulatory ß and γ subunits. We finish this review by describing how the association of the α subunit with ß or with γ subunits can change the BK channel phenotype and pharmacology.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio , Animales , HumanosRESUMEN
The dissipation of acute acid loads by the voltage-gated proton channel (Hv1) relies on regulating the channel's open probability by the voltage and the ΔpH across the membrane (ΔpH = pHex - pHin). Using monomeric Ciona-Hv1, we asked whether ΔpH-dependent gating is produced during the voltage sensor activation or permeation pathway opening. A leftward shift of the conductance-voltage (G-V) curve was produced at higher ΔpH values in the monomeric channel. Next, we measured the voltage sensor pH dependence in the absence of a functional permeation pathway by recording gating currents in the monomeric nonconducting D160N mutant. Increasing the ΔpH leftward shifted the gating charge-voltage (Q-V) curve, demonstrating that the ΔpH-dependent gating in Hv1 arises by modulating its voltage sensor. We fitted our data to a model that explicitly supposes the Hv1 voltage sensor free energy is a function of both the proton chemical and the electrical potential. The parameters obtained showed that around 60% of the free energy stored in the ΔpH is coupled to the Hv1 voltage sensor activation. Our results suggest that the molecular mechanism underlying the Hv1 ΔpH dependence is produced by protons, which alter the free-energy landscape around the voltage sensor domain. We propose that this alteration is produced by accessibility changes of the protons in the Hv1 voltage sensor during activation.
Asunto(s)
Algoritmos , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Modelos Biológicos , Protones , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/genética , Canales Iónicos/genética , Canales Iónicos/metabolismo , Potenciales de la Membrana/fisiología , Ratones , Simulación de Dinámica Molecular , Mutación , Oocitos/metabolismo , Oocitos/fisiología , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The majority of voltage-gated ion channels contain a defined voltage-sensing domain and a pore domain composed of highly conserved amino acid residues that confer electrical excitability via electromechanical coupling. In this sense, the voltage-gated proton channel (Hv1) is a unique protein in that voltage-sensing, proton permeation and pH-dependent modulation involve the same structural region. In fact, these processes synergistically work in concert, and it is difficult to separate them. To investigate the process of Hv1 voltage sensor trapping, we follow voltage-sensor movements directly by leveraging mutations that enable the measurement of Hv1 channel gating currents. We uncover that the process of voltage sensor displacement is due to two driving forces. The first reveals that mutations in the selectivity filter (D160) located in the S1 transmembrane interact with the voltage sensor. More hydrophobic amino acids increase the energy barrier for voltage sensor activation. On the other hand, the effect of positive charges near position 264 promotes the formation of salt bridges between the arginines of the voltage sensor domain, achieving a stable conformation over time. Our results suggest that the activation of the Hv1 voltage sensor is governed by electrostatic-hydrophobic interactions, and S4 arginines, N264 and selectivity filter (D160) are essential in the Ciona-Hv1 to understand the trapping of the voltage sensor.
Asunto(s)
Antifibrinolíticos , Ciona , Animales , Protones , Aminoácidos , ArgininaRESUMEN
The voltage-gated proton (Hv1) channel, a voltage sensor and a conductive pore contained in one structural module, plays important roles in many physiological processes. Voltage sensor movements can be directly detected by measuring gating currents, and a detailed characterization of Hv1 charge displacements during channel activation can help to understand the function of this channel. We succeeded in detecting gating currents in the monomeric form of the Ciona-Hv1 channel. To decrease proton currents and better separate gating currents from ion currents, we used the low-conducting Hv1 mutant N264R. Isolated ON-gating currents decayed at increasing rates with increasing membrane depolarization, and the amount of gating charges displaced saturates at high voltages. These are two hallmarks of currents arising from the movement of charged elements within the boundaries of the cell membrane. The kinetic analysis of gating currents revealed a complex time course of the ON-gating current characterized by two peaks and a marked Cole-Moore effect. Both features argue that the voltage sensor undergoes several voltage-dependent conformational changes during activation. However, most of the charge is displaced in a single central transition. Upon voltage sensor activation, the charge is trapped, and only a fast component that carries a small percentage of the total charge is observed in the OFF. We hypothesize that trapping is due to the presence of the arginine side chain in position 264, which acts as a blocking ion. We conclude that the movement of the voltage sensor must proceed through at least five states to account for our experimental data satisfactorily.
Asunto(s)
Ciona intestinalis/química , Ciona intestinalis/metabolismo , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Sustitución de Aminoácidos , Animales , Ciona intestinalis/genética , Canales Iónicos/genética , Transporte Iónico/fisiología , Cinética , Mutación Missense , Xenopus laevisRESUMEN
Two families of accessory proteins, ß and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Elementos de la Serie de los Lantanoides/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Subunidades de Proteína/química , Compuestos de Boro/químicaRESUMEN
The vancomycin loading dose (LD) of 25 to 30 mg/kg is a frequently practiced strategy to achieve effective concentrations from the first-treatment dose. However, considering only the body weight for dosing might be inadequate in critically ill patients due to pharmacokinetics changes. We sought to assess achieving optimal trough serum levels of vancomycin and AUC0-24/MIC in the first 24 h of treatment by using an LD based on population pharmacokinetic parameters of critically ill patients. We performed a concurrent cohort study over 22 months of patients with severe sepsis who received intravenous vancomycin. The patients were treated with three different strategies to initiate vancomycin: without an LD (group A), with an LD of 25 to 30 mg/kg (group B), and with an LD based on population pharmacokinetic parameters of the critically ill patient (group C). An optimal trough serum concentration was achieved in 5, 9, and 83% of patients in groups A, B, and C, respectively. The number of patients that reached optimal AUC0-24 was 2 of 18 (11%), 5 of 11 (46%), and 11 of 12 (92%) in groups A, B, and C, respectively. The statistical analysis for both parameters revealed significant differences in group C with respect to other groups. The administration of the LD calculated from population pharmacokinetic parameters from the beginning of therapy is a more efficient strategy to obtain adequate trough serum concentrations and AUC0-24/MIC in critical patients.
Asunto(s)
Antibacterianos/farmacocinética , Antibacterianos/uso terapéutico , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Vancomicina/farmacocinética , Vancomicina/uso terapéutico , Estudios de Cohortes , Cuidados Críticos/métodos , Enfermedad Crítica , Humanos , Sepsis/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Vancomicina/sangreRESUMEN
Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales Catiónicos TRPM/fisiología , Temperatura , Algoritmos , Animales , Frío , Estimulación Eléctrica , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mentol/farmacología , Modelos Biológicos , Oocitos/metabolismo , Oocitos/fisiología , Fosfatidilinositol 4,5-Difosfato/farmacología , Ratas , Canales Catiónicos TRPM/genética , Xenopus laevisRESUMEN
Copper, an ion with many important metabolic functions, has also been proposed to have a role as modulator on neuronal function, mostly based on its effects on voltage- and neurotransmitter-gated conductance as well as on neurological symptoms of patients with altered copper homeostasis. Nevertheless, the mechanisms by which copper exerts its neuromodulatory effects have not been clearly established in a functional neuronal network. Using rat hippocampus slices as a neuronal network model, the effects of copper in the range of 10-100 nm were tested on the intrinsic, synaptic and network properties of the CA1 region. Most of the previously described effects of this cation were in the micromolar range of copper concentrations. The current results indicate that copper is a multifaceted neuromodulator, having effects that may be grouped into two categories: (i) activity enhancement, by modulating synaptic communication and action potential (AP) conductances; and (ii) temporal processing and correlation extraction, by improving reliability and depressing inhibition. Specifically it was found that copper hyperpolarizes AP firing threshold, enhances neuronal and network excitability, modifies CA3-CA1 pathway gain, enhances the frequency of spontaneous synaptic events, decreases inhibitory network activity, and improves AP timing reliability. Moreover, copper chelation by bathocuproine decreases spontaneous network spiking activity. These results allow the proposal that copper affects the network activity from cellular to circuit levels on a moment-by-moment basis, and should be considered a crucial functional component of hippocampal neuronal circuitry.
Asunto(s)
Cobre/metabolismo , Hipocampo/fisiología , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Quelantes/farmacología , Simulación por Computador , Cobre/administración & dosificación , Relación Dosis-Respuesta a Droga , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/efectos de los fármacos , Microelectrodos , Modelos Neurológicos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Fenantrolinas/farmacología , Ratas Sprague-Dawley , Canales de Sodio/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms converge to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming α subunit of BK is associated with one of four alternative ß subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, ß1 stabilizes the active configuration of the BK voltage sensor having a large effect on BK Ca(2+) sensitivity. To determine the extent to which ß subunits regulate the BK voltage sensor, we measured gating currents induced by the pore-forming BK α subunit alone and with the different ß subunits expressed in Xenopus oocytes (ß1, ß2IR, ß3b, and ß4). We found that ß1, ß2, and ß4 stabilize the BK voltage sensor in the active conformation. ß3 has no effect on voltage sensor equilibrium. In addition, ß4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in α ß4 channels explains most of their biophysical properties. For channels composed of the α subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of this charge develops with a time course comparable to that of K(+) current activation. In the presence of ß1, ß2, and ß4 this slow component develops in advance of and much more rapidly than ion current activation, suggesting that BK channel opening proceeds in two steps.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Subunidades de Proteína/metabolismo , Regulación Alostérica/fisiología , Animales , Calcio/metabolismo , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Oocitos/citología , Oocitos/metabolismo , Potasio/metabolismo , Subunidades de Proteína/genética , Xenopus laevisRESUMEN
In this chapter we discuss the polymodal activation of thermo-TRP channels using as exemplars two of the best characterized members of this class of channels: TRPM8 and TRPV1. Since channel activation by temperature is the hallmark of thermo-TRP channels, we present a detailed discussion on the thermodynamics involved in the gating processes by temperature, voltage, and agonists. We also review recently published data in an effort to put together all the pieces available of the amazing puzzle of thermo-TRP channel activation. Special emphasis is made in the structural components that allow the channel-forming proteins to integrate such diverse stimuli, and in the coupling between the different sensors and the ion conduction pathway. We conclude that the present data is most economically explained by allosteric models in which temperature, voltage, and agonists act separately to modulate channel activity.
Asunto(s)
Receptores de Superficie Celular/fisiología , Canales de Potencial de Receptor Transitorio/fisiología , Regulación Alostérica , Secuencia de Aminoácidos , Biofisica , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Temperatura , Canales de Potencial de Receptor Transitorio/agonistasRESUMEN
BACKGROUND: Malignant pleural mesothelioma (MPM) is rare and aggressive neoplasia, with a poor prognosis; furthermore, the monetary cost of its treatment represents a major challenge for many patients. The economic burden this malignancy imposes is underscored by the fact that asbestos exposure, which is the most frequent risk factor, is much more prevalent in the lower socioeconomic population of developing countries. The aims of the present study were to evaluate the efficacy, safety, and cost of continuous infusion of low-dose Gemcitabine plus Cisplatin (CIGC) as a treatment strategy for patients with unresectable MPM. METHODS: We performed a prospective cohort study to determine efficacy and safety of continuous infusion gemcitabine at a dose of 250 mg/m2 in a 6-h continuous infusion plus cisplatin 35 mg/m2 on days 1 and 8 of a 21-day cycle in patients with unresectable MPM. We also performed a cost-minimization analysis to determine if this chemotherapy regimen is less expensive than other currently used regimens. RESULTS: The median number of chemotherapy cycles was six (range 1-11 cycles); objective response rate was documented in 46.2%, and disease control rate was seen in 81.2%. Median PFS was 8.05 months (CI 95% 6.97-9.13); median OS was 16.16 months (CI 95% 12.5-19.9). The cost minimization analysis revealed savings of 66.4, 61.9, and 97.7% comparing CIGC with short-infusion gemcitabine plus cisplatin (SIGC), cisplatin plus pemetrexed (CP), and cisplatin plus pemetrexed and bevacizumab (CPB), respectively. Furthermore, this chemotherapy regimen proved to be safe at the administered dosage. CONCLUSION: CIGC is an effective and safe treatment option for patients with unresectable MPM; besides, this combination is a cost-saving option when compared with other frequently used chemotherapy schemes. Therefore, this treatment scheme should be strongly considered for patients with unresectable MPM and limited economic resources.
RESUMEN
The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Mutación , Algoritmos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Bario/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Caribdotoxina/farmacología , Simulación por Computador , Femenino , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potenciales de la Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Concentración Osmolar , Técnicas de Placa-Clamp , Potasio/metabolismo , Potasio/farmacología , ARN Complementario/administración & dosificación , ARN Complementario/genética , Homología de Secuencia de Aminoácido , Electricidad Estática , Tetraetilamonio/farmacología , Xenopus laevisRESUMEN
A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide range of voltages, temperatures, and agonist concentrations. We have achieved this in the case of the TRPM8 channel expressed in Xenopus laevis oocytes. Measurements covered an extensive range of probabilities and unprecedented applied voltages up to 500 mV. In this chapter, we describe animal care protocols of patch-clamp pipette preparation, temperature control methods, and analysis of ionic currents to obtain reliable absolute open channel probabilities.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potencial de Receptor Transitorio/fisiología , Xenopus laevis , Animales , Electrofisiología/métodos , Activación del Canal Iónico/genética , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Probabilidad , Temperatura , Xenopus laevis/fisiología , Xenopus laevis/cirugíaRESUMEN
Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.
Asunto(s)
Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Cationes Bivalentes/metabolismo , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
Mutations in connexin 26 (Cx26) hemichannels can lead to syndromic deafness that affects the cochlea and skin. These mutations lead to gain-of-function hemichannel phenotypes by unknown molecular mechanisms. In this study, we investigate the biophysical properties of the syndromic mutant Cx26G12R (G12R). Unlike wild-type Cx26, G12R macroscopic hemichannel currents do not saturate upon depolarization, and deactivation is faster during hyperpolarization, suggesting that these channels have impaired fast and slow gating. Single G12R hemichannels show a large increase in open probability, and transitions to the subconductance state are rare and short-lived, demonstrating an inoperative fast gating mechanism. Molecular dynamics simulations indicate that G12R causes a displacement of the N terminus toward the cytoplasm, favoring an interaction between R12 in the N terminus and R99 in the intracellular loop. Disruption of this interaction recovers the fast and slow voltage-dependent gating mechanisms. These results suggest that the mechanisms of fast and slow gating in connexin hemichannels are coupled and provide a molecular mechanism for the gain-of-function phenotype displayed by the syndromic G12R mutation.
Asunto(s)
Conexina 26/metabolismo , Sordera/genética , Ictiosis/genética , Activación del Canal Iónico , Queratitis/genética , Mutación Missense , Animales , Conexina 26/química , Conexina 26/genética , Humanos , Simulación de Dinámica Molecular , XenopusRESUMEN
In 1943, David Goldman published a seminal paper in The Journal of General Physiology that reported a concise expression for the membrane current as a function of ion concentrations and voltage. This body of work was, and still is, the theoretical pillar used to interpret the relationship between a cell's membrane potential and its external and/or internal ionic composition. Here, we describe from an historical perspective the theory underlying the constant-field equation and its application to membrane ion transport.
Asunto(s)
Membrana Celular/metabolismo , Electrofisiología/historia , Canales Iónicos/metabolismo , Animales , Membrana Celular/fisiología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Transporte IónicoRESUMEN
Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.
Asunto(s)
Arabidopsis/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Algoritmos , Proteínas de Arabidopsis , ADN Complementario/biosíntesis , ADN Complementario/genética , Electrofisiología , Mesilatos , Técnicas de Placa-Clamp , Proteínas de Plantas , Compuestos de Amonio Cuaternario , ARN Mensajero/biosíntesisRESUMEN
Mammals maintain homeostatic control of their body temperature. Therefore, these organisms are expected to have adaptations that confer the ability to detect and react to both self and ambient temperature. Temperature-activated ion channels have been discovered to be the primary molecular determinants of thermosensation. The most representative group of these determinants constitutes members of the transient receptor potential superfamily, TRP, which are activated by either low or high temperatures covering the whole range of physiologically relevant temperatures. This review makes a critical assessment of existing analytical methods of temperature-activated TRP channel mechanisms using the cold-activated TRPM8 channel as a paradigm.