RESUMO
The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.
Assuntos
Actinina/metabolismo , Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Actinina/genética , Substituição de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Calmodulina/genética , Humanos , Mutação , Neurônios/metabolismo , Ligação ProteicaRESUMO
The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327-357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4.
Assuntos
Ativação do Canal Iônico , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Carbocianinas/química , Cisteína/química , Cisteína/genética , Ligação de Hidrogênio , Ativação do Canal Iônico/genética , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Relação Estrutura-AtividadeRESUMO
KEY POINTS: The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. ABSTRACT: Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+ -dependent Cl- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+ ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+ ]o from 10-10 to 10-5.5 m caused a progressive increase in the chloride current (ICl ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+ ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10-5.5 and 10-9.0 m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5 m, but 10-9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+ ]i .
Assuntos
Canais de Cloreto/fisiologia , Anoctamina-1 , Cálcio/fisiologia , Canais de Cloreto/genética , Células HEK293 , Humanos , Modelos Moleculares , PrótonsRESUMO
Neuronal M-type K(+) channels are heteromers of KCNQ2 and KCNQ3 subunits, and are found in cell bodies, dendrites and the axon initial segment, regulating the firing properties of neurons. By contrast, presynaptic KCNQ2 homomeric channels directly regulate neurotransmitter release. Previously, we have described a mechanism for gating downregulation of KCNQ2 homomeric channels by calmodulin and syntaxin1A. Here, we describe a new mechanism for regulation of KCNQ2 channel gating that is modulated by Src, a non-receptor tyrosine kinase. In this mechanism, two concurrent distinct structural rearrangements of the cytosolic termini induce two opposing effects: upregulation of the single-channel open probability, mediated by an N-terminal tyrosine, and reduction in functional channels, mediated by a C-terminal tyrosine. In contrast, Src-mediated regulation of KCNQ3 homomeric channels, shown previously to be achieved through the corresponding tyrosine residues, involves the N-terminal-tyrosine-mediated downregulation of the open probability, rather than an upregulation. We argue that the dual bidirectional regulation of KCNQ2 functionality by Src, mediated through two separate sites, means that KCNQ2 can be modified by cellular factors that might specifically interact with either one of the sites, with potential significance in the fine-tuning of neurotransmitters release at nerve terminals.
Assuntos
Ativação do Canal Iônico , Canal de Potássio KCNQ2/fisiologia , Neurônios/fisiologia , Transmissão Sináptica , Animais , Axônios/fisiologia , Calmodulina/metabolismo , Citosol/fisiologia , Humanos , Proteínas Recombinantes/metabolismo , Xenopus laevis , Quinases da Família src/fisiologiaRESUMO
This perspective attempts to shed light on an old and not yet solved controversy in cardiac physiology, i.e., the impact of increasing ryanodine receptor (RyR)2 open probability on myocardial function. Based on an already proven myocyte model, it was shown that increasing RyR2 open probability results in a purely short-lived increase in Ca2+ transient amplitude, and, therefore, it does not increase cardiac contractility. However, potentiation of RyR2 activity permanently enhances fractional Ca2+ release, shifting the intracellular Ca2+ transient versus sarcoplasmic reticulum (SR) Ca2+ content curve to a new state of higher efficiency. This would allow the heart to maintain a given contractility despite a decrease in SR Ca2+ content, to enhance contractility if SR Ca2+ content is simultaneously preserved or to successfully counteract the effects of a negative inotropic intervention.NEW & NOTEWORTHY Increasing ryanodine receptor (RyR)2 open probability does not increase cardiac contractility. However, RyR2 potentiation shifts the intracellular Ca2+ transient-sarcoplasmic reticulum (SR) Ca2+ content relationship toward an enhanced efficiency state, which may contribute to a positive inotropic effect, preserve contractility despite decreased SR Ca2+ content, or successfully counteract the effects of a negative inotropic action.
Assuntos
Sinalização do Cálcio , Ativação do Canal Iônico , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Humanos , Cinética , Potenciais da Membrana , Modelos Cardiovasculares , Retículo Sarcoplasmático/metabolismoRESUMO
All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage sensitivity for smaller populations of channels by reducing the amount of sphingomyelin in the membrane. Given lysenin's preference for targeting lipid rafts, this result indicates the potential role of the heterogeneous organization of the membrane in modulating channel functionality. Our work indicates that local congestion within membranes may alter the energy landscape and the kinetics of conformational changes of lysenin channels in response to voltage stimuli. This level of understanding may be extended to better characterize the role of the specific membrane environment in modulating the biological functionality of protein channels in health and disease.
Assuntos
Ativação do Canal Iônico , Microdomínios da Membrana/química , Potenciais da Membrana , Toxinas Biológicas/química , Bicamadas Lipídicas/química , Microdomínios da Membrana/metabolismo , Esfingomielinas/química , Toxinas Biológicas/metabolismoRESUMO
This study aimed to investigate the intracellular Mg(2+) regulation of the L-type Ca(2+) channels in guinea pig ventricular myocytes. By adopting the inside-out configuration of the patch clamp technique, single channel currents of the L-type Ca(2+) channels were recorded at different intracellular Mg(2+) concentrations ([Mg(2+)]i). At free [Mg(2+)]i of 0, 10(-9), 10(-7), 10(-5), 10(-3), and 10(-1) M, 1.4 µM CaM + 3 mM ATP induced channel activities of 44%, 117%, 202%, 181%, 147%, and 20% of the control activity in cell-attached mode, respectively, showing a bell-shaped concentration-response relationship. Moreover, the intracellular Mg(2+) modulated the Ca(2+) channel gating properties, accounting for alterations in channel activities. These results imply that Mg(2+) has a dual effect on the L-type Ca(2+) channels: facilitation and inhibition. Lower [Mg(2+)]i maintains and enhances the basal activity of Ca(2+) channels, whereas higher [Mg(2+)]i inhibits channel activity. Taken together, our data from the application of an [Mg(2+)]i series suggest that the dual effect of Mg(2+) upon the L-type Ca(2+) channels exhibits long open-time dependence.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Magnésio/fisiologia , Células Musculares/metabolismo , Animais , Células Cultivadas , Cobaias , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Magnésio/farmacologia , Técnicas de Patch-Clamp/métodosRESUMO
Epithelial Na(+) channel (ENaC) subunits (α, ß, and γ) found in functional complexes are translated from mature mRNAs that are similarly processed by the inclusion of 13 canonical exons. We examined whether individual exons 3-12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α-subunit exon were expressed in Xenopus oocytes, and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α-subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited fourfold greater amiloride-sensitive whole cell currents than cells expressing wild-type channels. A parallel fivefold increase in channel open probability was observed with channels lacking α-subunit exon 11. These mutant channels also exhibited a lost of Na(+) self-inhibition, whereas we found similar levels of surface expression of mutant and wild-type channels. In contrast, in-frame deletions of exon 11 from either the ß- or γ-subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encodes structurally homologous yet functionally diverse domains and that exon 11 in the α-subunit encodes a module that regulates channel gating.
Assuntos
Canais Epiteliais de Sódio/genética , Deleção de Sequência , Xenopus laevis/metabolismo , Amilorida/farmacologia , Animais , Células Cultivadas , Canais Epiteliais de Sódio/metabolismo , Éxons , Humanos , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Mutação Puntual/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Deleção de Sequência/efeitos dos fármacos , Xenopus laevis/genéticaRESUMO
Ca(2+)-activated Cl(-) channels (CaCCs) are anion-selective channels and involved in physiological processes such as electrolyte/fluid secretion, smooth muscle excitability, and olfactory perception which critically depend on the Ca(2+) and voltage dual-dependent gating of channels. However, how the Ca(2+) and voltage regulate the gating of CaCCs still unclear. In this work, the authors constructed a biophysical model to illustrate the dual-dependent gating of CaCCs. For validation, we applied our model on both native CaCCs and exogenous TMEM16A which is thought to be the molecular basis of CaCCs. Our data show that the native CaCCs may share universal gating mechanism. We confirmed the assumption that by binding with the channel, Ca(2+) decreases the energy-barrier to open the channel, but not changes the voltage-sensitivity. For TMEM16A, our model indicates that the exogenous channels show different Ca(2+) dependent gating mechanism from the native ones. These results advance the understanding of intracellular Ca(2+) and membrane potential regulation in CaCCs, and shed new light on its function in aspect of physiology and pharmacology.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Anoctamina-1 , Cálcio/química , Canais de Cloreto/química , Humanos , Proteínas de Neoplasias/químicaRESUMO
The epithelial sodium channel (ENaC) is comprised of three homologous subunits. Channels composed solely of α- and ß-subunits (αß-channels) exhibit a very high open probability (Po) and reduced sensitivity to amiloride, in contrast to channels composed of α- and γ-subunits or of all three subunits (i.e., αγ- and αßγ-channels). A mutant channel comprised of α- and ß-subunits, and a chimeric γ-subunit where the region immediately preceding (ß12 and wrist) and encompassing the second transmembrane domain (TM2) was replaced with the corresponding region of the ß-subunit (γ-ßTM2), displayed characteristics reminiscent of αß-channels, including a reduced amiloride potency of block and a loss of Na(+) self-inhibition (reflecting an increased Po). Substitutions at key pore-lining residues of the γ-ßTM2 chimera enhanced the Na(+) self-inhibition response, whereas key γ-subunit substitutions reduced the response. Furthermore, multiple sites within the TM2 domain of the γ-subunit were required to confer high amiloride potency. In summary, we have identified novel pore-lining residues of the γ-subunit of ENaC that are important for proper channel gating and its interaction with amiloride.
Assuntos
Amilorida/farmacologia , Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico , Transporte de Íons/efeitos dos fármacos , Mutação/genética , Sequência de Aminoácidos , Animais , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Transporte de Íons/genética , Camundongos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , XenopusRESUMO
Ryanodine receptors are generally thought to possess a high-affinity activating cytosolic Ca(2+) site and a low-affinity inhibitory cytosolic Ca(2+) site. By performing conformation selective measurements in which quercetin was used as a fluorescent marker for RyR3 (ryanodine receptor type 3) in Jurkat cells, we now find that the rectified RyR3 channel in open conformation may be regulated in situ by two cytosolic activating Ca(2+) sites, of low and high affinity, respectively, whereas no inhibitory Ca(2+) effect could be delineated. In the closed rectified channel, as well as in the open hindered channel, only the high affinity activating Ca(2+) site and the inhibitory Ca(2+) site were functional, whereas in the closed hindered channel all three regulatory Ca(2+) sites appeared to be operational. RyR3 also seems to possess one activating and two inhibitory quercetin sites. Corresponding Hill coefficients and affinities of these regulatory sites were estimated. Quercetin cellular uptake exhibited an initial rapid phase (~1.04 min), followed by slow accumulation of free quercetin inside the cytosol (~34.5 min). The RyR3-mediated Ca(2+) release current increased from a baseline of 247 to 287 pA in 1 min. after addition of 50 µM quercetin and then declined slowly to a plateau of 265 pA.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Corantes Fluorescentes/metabolismo , Quercetina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Citosol/metabolismo , Humanos , Ativação do Canal Iônico , Células Jurkat , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Espectrometria de FluorescênciaRESUMO
BACKGROUND: L-type CaV1.2 calcium channel, the primary gateway for Ca2+ influx in smooth muscles, is widely regulated by multiple posttranslational modifications, such as protein kinase-mediated phosphorylation and nitric oxide-induced S-nitrosylation. However, the effect of S-nitrosylation on CaV1.2 channel function and its role in arterial contractility are not well understood. METHODS: Electrophysiological recordings, Ca2+ and confocal imaging, and biochemical assays were used to functionally characterize S-nitrosylated CaV1.2 channels in vitro, while pressure myography and tail-cuff blood pressure measurement were conducted to evaluate the physiological effects of CaV1.2 S-nitrosylation ex vivo and in vivo. RESULTS: S-nitrosylation significantly reduced the CaV1.2 current density by promoting lysosomal degradation that leads to decreased levels of total and surface CaV1.2 channel proteins in a CaVß-independent manner and reducing the open probability of CaV1.2 channel. Mechanistically, the Cys1180 and Cys1280 residues within CaV1.2 channel have been determined as the molecular targets for S-nitrosylation as substitution of either Cys1180 or Cys1280 for serine resulted in substantial reduction of S-nitrosylation levels. Of note, CaV1.2 S-nitrosylation levels were significantly reduced in arteries isolated from both spontaneously hypertensive rats and patients with pulmonary hypertension. Moreover, mouse resistance arteries incubated with S-nitrosocysteine displayed much lower contractility and spontaneously hypertensive rats injected with S-nitrosocysteine also showed significantly reduced blood pressure, suggesting that reduced S-nitrosylation contributes to the upregulation of CaV1.2 channel activity in hypertensive arteries. CONCLUSIONS: This study provides strong evidence that S-nitrosylation-mediated downregulation of CaV1.2 channels is via 2 distinctive mechanisms and the findings offer potential pathways for therapeutic inventions in hypertension.
Assuntos
Hipertensão , Vasoconstrição , Ratos , Camundongos , Animais , Ratos Endogâmicos SHR , Óxido Nítrico/metabolismo , Músculo Liso Vascular/metabolismo , Canais de Cálcio Tipo L/metabolismo , ProbabilidadeRESUMO
The function of ion channels to mediate the flux of ions through membranes of living cells depends on their number, conductance, and open probability. The open probability, PO, characterizes gating of channels that is sensitive to experimental conditions and that can be determined in single-channel experiments. Individual experimental records and even whole series of single-channel activity measurements represent random samples of the stochastic gating continuous in time. The aim of this study was to understand the relationship between the accuracy (trueness and precision) of PO determination and the method of single-channel activity data collection. We used simulated single-channel experiments with variable settings of data collection for a range of open probability values. We found that at low PO, the trueness of PO determination depends on the average number of channel openings per record, while the precision of PO determination depends on the total number of channel openings in the whole dataset and on the distribution of open and closed times. We derived relationships that allow planning of single-channel experiments for the required accuracy of PO determination over a large span of open probabilities.
Assuntos
Eletrofisiologia/métodos , Ativação do Canal Iônico , Canais Iônicos/fisiologia , Animais , Humanos , Cinética , Potenciais da Membrana , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Probabilidade , Reprodutibilidade dos TestesRESUMO
Action potentials arriving at a nerve terminal activate voltage-gated calcium channels and set the electrical driving force for calcium entry which affects the amount and duration of neurotransmitter release. During propagation, the duration, amplitude, and shape of action potentials often changes. This affects calcium entry, and can cause large changes in neurotransmitter release. Here, we have used a series of amplitude and area matched stimuli to examine how the shape and amplitude of a stimulus affect calcium influx at a presynaptic nerve terminal in the mammalian brain. We identify fundamental differences in calcium entry following calcium channel activation by a standard voltage jump vs. an action potential-like stimulation. We also tested a series of action potential-like stimuli with the same amplitude, duration, and stimulus area, but differing in their rise and decay times. We find that a stimulus that matches the rise and decay times of a physiological action potential produces a calcium channel response that is optimized over a range of peak amplitudes. Next, we determined the relative number of calcium channels that are active at different times during an action potential, which is important in the context of how local calcium domains trigger neurotransmitter release. We find the depolarizing phase of an AP-like stimulus only opens ~20% of the maximum number of calcium channels that can be activated. Channels continue to activate during the falling phase of the action potential, with peak calcium channel activation occurring near 0 mV. Although less than 25% of calcium channels are active at the end of the action potential, these calcium channels will generate a larger local calcium concentration that will increase the release probability for nearby vesicles. Determining the change in open probability of presynaptic calcium channels, and taking into account how local calcium concentration also changes throughout the action potential are both necessary to fully understand how the action potential triggers neurotransmitter release.
RESUMO
Atomic resolution structures have provided significant insight into the gating and permeation mechanisms of various ion channels, including potassium channels. However, ion channels may also be regulated by numerous factors, including the physiochemical properties of the membrane in which they are embedded. For example, the matching of the bilayer's hydrophobic region to the hydrophobic external surface of the ion channel is thought to minimize the energetic penalty needed to solvate hydrophobic residues or exposed lipid tails. To understand the molecular basis of such regulation by hydrophobic matching requires examining channels in the presence of the lipid membrane. Here we examine the role of hydrophobic matching in regulating the activity of the model potassium channel, KcsA. 86Rb+ influx assays and single-channel recordings indicate that the non-inactivating E71A KcsA channel is most active in thin bilayers (Assuntos
Proteínas de Bactérias/metabolismo
, Membrana Celular/química
, Canais de Potássio/metabolismo
, Streptomyces lividans/metabolismo
, Substituição de Aminoácidos
, Proteínas de Bactérias/genética
, Membrana Celular/genética
, Membrana Celular/metabolismo
, Simulação de Dinâmica Molecular
, Mutação de Sentido Incorreto
, Canais de Potássio/genética
, Estrutura Secundária de Proteína
, Streptomyces lividans/genética
RESUMO
N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca2+-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing.
RESUMO
AIM: The L-type voltage-gated calcium channel Cav1.2 mediates the calcium influx into cells upon membrane depolarization. The list of cardiopathies associated to Cav1.2 dysfunctions highlights the importance of this channel in cardiac physiology. Calcium/calmodulin-dependent serine protein kinase (CASK), expressed in cardiac cells, has been identified as a regulator of Cav2.2 channels in neurons, but no experiments have been performed to investigate its role in Cav1.2 regulation. METHODS AND RESULTS: Full length or the distal C-terminal truncated of the pore-forming Cav1.2 channel (Cav1.2α1c), both present in cardiac cells, were expressed in TsA-201 cells. In addition, a shRNA silencer, or scramble as negative control, of CASK was co-transfected in order to silence CASK endogenously expressed. Three days post-transfection, the barium current was increased only for the truncated form without alteration of the steady state activation and inactivation biophysical properties. The calcium current, however, was increased after CASK silencing with both types of Cav1.2α1c subunits suggesting that, in absence of calcium, the distal C-terminal counteracts the CASK effect. Biochemistry experiments did not reveals neither an alteration of Cav1.2 channel protein expression after CASK silencing nor an interaction between Cav1.2α1c subunits and CASK. Nevertheless, after CASK silencing, single calcium channel recordings have shown an increase of the voltage-gated calcium channel Cav1.2 open probability explaining the increase of the whole-cell current. CONCLUSION: This study suggests CASK as a novel regulator of Cav1.2 via a modulation of the voltage-gated calcium channel Cav1.2 open probability.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Guanilato Quinases/metabolismo , HumanosRESUMO
During development of the central nervous system, there is a shift in the subunit composition of NMDA receptors (NMDARs) resulting in a dramatic acceleration of NMDAR-mediated synaptic currents. This shift coincides with upregulation of the GluN2A subunit and triheteromeric GluN1/2A/2B receptors with fast deactivation kinetics, whereas expression of diheteromeric GluN1/2B receptors with slower deactivation kinetics is decreased. Here, we show that allosteric interactions occur between the glutamate-binding GluN2 subunits in triheteromeric GluN1/2A/2B NMDARs. This allosterism is dominated by the GluN2A subunit and results in functional properties not predicted by those of diheteromeric GluN1/2A and GluN1/2B NMDARs. These findings suggest that GluN1/2A/2B NMDARs may maintain some signaling properties of the GluN2B subunit while having the kinetic properties of GluN1/2A NMDARs and highlight the complexity in NMDAR signaling created by diversity in subunit composition.
Assuntos
Regulação Alostérica/genética , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animais , Retículo Endoplasmático , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Plasticidade Neuronal , Técnicas de Patch-Clamp , Subunidades Proteicas , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica , Xenopus laevisRESUMO
The effects of noise on the calcium oscillations in a cell exposed to electromagnetic fields are described by a dynamic model. Noise is a very important factor to be considered in the dynamic research on the calcium oscillations in a cell exposed to electromagnetic fields. Some meaningful results have been obtained here based on the discussion. The results show that the pattern of intracellular calcium oscillations exposure to electromagnetic fields can be influenced by noise. Furthermore, the intracellular calcium oscillations exposure to electromagnetic fields can also be induced by noise. And the work has also studied the relationships between the voltage sensitive calcium channel's open probability and electromagnetic field. The result can provide new insights into constructive roles and potential applications of selecting appropriate electromagnetic field frequency during the research of biological effect of electromagnetic field.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Citosol/metabolismo , Animais , Simulação por Computador , Campos Eletromagnéticos , Humanos , Modelos Biológicos , ProbabilidadeRESUMO
Intracellular [Na(+)] ([Na(+)]i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na(+) transport, but the underlying mechanisms remain unclear. We show here that short-term (60-80 min) incubation of ENaC-expressing oocytes in high Na(+) results in a 75% decrease in channel activity. When the ß subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na(+)]i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na(+) reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na(+) incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na(+) and 4 hrs in high Na(+), suggesting that [Na(+)]i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na(+)]i.