RESUMO
The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Ligantes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , AMP Cíclico/metabolismo , Fenômenos Biofísicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismoRESUMO
Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four α-subunits, each with six transmembrane segments (S1-S6). Silent Kv (KvS), includes Kv5.1, Kv6.1-6.4, Kv8.1-8.2, and Kv9.1-9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4-S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology.
RESUMO
Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of â¼-3.5 to â¼-5.5 kJ â mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of â¼-9.6 to â¼-9.9 kJ â mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.
Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação do Canal Iônico , Termodinâmica , Animais , Sítios de Ligação , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Ligantes , Oócitos/metabolismo , Conformação Proteica , Subunidades Proteicas , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
Cyclic nucleotide-gated (CNG) ion channels of olfactory sensory neurons contain three types of homologue subunits, two CNGA2 subunits, one CNGA4 subunit and one CNGB1b subunit. Each subunit carries an intracellular cyclic nucleotide binding domain (CNBD) whose occupation by up to four cyclic nucleotides evokes channel activation. Thereby, the subunits interact in a cooperative fashion. Here we studied 16 concatamers with systematically disabled, but still functional, binding sites and quantified channel activation by systems of intimately coupled state models transferred to 4D hypercubes, thereby exploiting a weak voltage dependence of the channels. We provide the complete landscape of free energies for the complex activation process of heterotetrameric channels, including 32 binding steps, in both the closed and open channel, as well as 16 closed-open isomerizations. The binding steps are specific for the subunits and show pronounced positive cooperativity for the binding of the second and the third ligand. The energetics of the closed-open isomerizations were disassembled to elementary subunit promotion energies for channel opening, [Formula: see text], adding to the free energy of the closed-open isomerization of the empty channel, E0. The [Formula: see text] values are specific for the four subunits and presumably invariant for the specific patterns of liganding. In conclusion, subunit cooperativity is confined to the CNBD whereas the subunit promotion energies for channel opening are independent.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos , Neurônios Receptores Olfatórios , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ligantes , Nucleotídeos Cíclicos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , OlfatoRESUMO
P2X receptors are trimeric nonselective cation channels gated by ATP. They assemble from seven distinct subunit isoforms as either homo- or heteromeric complexes and contain three extracellularly located binding sites for ATP. P2X receptors are expressed in nearly all tissues and are there involved in physiological processes like synaptic transmission, pain, and inflammation. Thus, they are a challenging pharmacological target. The determination of crystal and cryo-EM structures of several isoforms in the last decade in closed, open, and desensitized states has provided a firm basis for interpreting the huge amount of functional and biochemical data. Electrophysiological characterization in conjugation with optical approaches has generated significant insights into structure-function relationships of P2X receptors. This review focuses on novel optical and related approaches to better understand the conformational changes underlying the activation of these receptors.
Assuntos
Trifosfato de Adenosina , Ativação do Canal Iônico , Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2X/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within â¼1 ms, while all other receptors are thought to become activated within â¼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 â¼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 â¼ 20 ms). Receptor deactivation occurred with time constants of â¼40 and â¼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Dimerização , Células HEK293 , Humanos , Cinética , Receptores Acoplados a Proteínas G/efeitos da radiaçãoRESUMO
Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker (CL) disk. Confocal patch-clamp fluorometry revealed pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL-CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity-theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP-binding sites or toward the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP-binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Animais , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , Nucleotídeos Cíclicos , Ligação ProteicaRESUMO
P2X7 receptors are trimeric ion channels activated by extracellular ATP. Upon activation, they trigger cytolysis and apoptosis but also control cell proliferation. To shed more light on channel gating and the underlying function of the individual subunits, receptors of concatenated subunits were built containing a defined number of functional binding sites. The currents evoked by ATP were obtained in the outside-out configuration of the patch-clamp technique, and steady-state activation, as well as time courses, were analyzed. Our results show that each occupied binding site contributes to channel activation. While the occupation of a single binding site can already activate the channels, three bound ligands maximally stabilize the open state. Hence, P2X7 receptors can be described by a stepwise activation process.
Assuntos
Trifosfato de Adenosina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mutação de Sentido Incorreto , Oócitos/fisiologia , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Sítios de Ligação/genética , Feminino , Ativação do Canal Iônico/genética , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Fatores de Tempo , Xenopus laevisRESUMO
Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.
Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Agonistas do Receptor Purinérgico P2X/síntese química , Agonistas do Receptor Purinérgico P2X/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Ligantes , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
High-affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools for investigating their natural targets. Cyclic nucleotide-regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging, and there is a need for fluorescent probes that enable the process to be broken down to the single-molecule level. This inspired us to prepare fluorophore-labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. These bioconjugates were prepared by a novel cross-linking strategy that involves substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide-regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation-π interactions between the ligand and the protein. Moreover, the introduced substances have the potential to investigate related target proteins, such as cAMP- and cGMP-dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases.
Assuntos
AMP Cíclico/química , AMP Cíclico/farmacologia , GMP Cíclico/química , GMP Cíclico/farmacologia , Corantes Fluorescentes/química , Canais Iônicos/agonistas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.
Assuntos
AMP Cíclico/química , GMP Cíclico/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Corantes Fluorescentes/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Imagem Individual de MoléculaRESUMO
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control electrical rhythmicity in specialized brain and heart cells. We quantitatively analysed voltage-dependent activation of homotetrameric HCN2 channels and its modulation by the second messenger cAMP using global fits of hidden Markovian models to complex experimental data. We show that voltage-dependent activation is essentially governed by two separable voltage-dependent steps followed by voltage-independent opening of the pore. According to this model analysis, the binding of cAMP to the channels exerts multiple effects on the voltage-dependent gating: It stabilizes the open pore, reduces the total gating charge from ~8 to ~5, makes an additional closed state outside the activation pathway accessible and strongly accelerates the ON-gating but not the OFF-gating. Furthermore, the open channel has a much slower computed OFF-gating current than the closed channel, in both the absence and presence of cAMP. Together, these results provide detailed new insight into the voltage- and cAMP-induced activation gating of HCN channels.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Ativação do Canal Iônico/fisiologia , Animais , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Cinética , Cadeias de Markov , Modelos Neurológicos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Xenopus laevis/fisiologiaRESUMO
Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3',5'-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.
Assuntos
AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Adenina/análogos & derivados , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Animais , AMP Cíclico/síntese química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/agonistas , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Halogenação , Humanos , CamundongosRESUMO
Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , Família Multigênica/genética , Animais , Clonagem Molecular , AMP Cíclico/metabolismo , Microscopia Confocal , Oócitos/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Homologia de Sequência , XenopusRESUMO
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3',5'-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels not preactivated by voltage. We identified a conformational flip of the channel as an intermediate state following the ligand binding and quantified it kinetically. Globally fitting the time courses of ligand binding and unbinding revealed modest cooperativity among the subunits in the conformational flip. The intensity of this cooperativity, however, was only moderate compared to channels preactivated by hyperpolarizing voltage. These data provide kinetic information about conformational changes proceeding in nonactivated HCN2 channels when cAMP binds. Moreover, our approach bears potential for analyzing the function of any other membrane receptor if a potent fluorescent ligand is available.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Nucleotídeos Cíclicos/farmacologia , Subunidades Proteicas/química , Animais , AMP Cíclico/metabolismo , Feminino , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Conformação Proteica , Subunidades Proteicas/metabolismo , Xenopus laevisRESUMO
P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,ßMe-ATP (α,ßMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.
Assuntos
Trifosfato de Adenosina , Receptores Purinérgicos P2X , Humanos , Ligantes , Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2X/química , Magnésio/metabolismo , Magnésio/química , Ligação Proteica , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X3/química , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X2/química , Agonistas do Receptor Purinérgico P2X/farmacologiaRESUMO
Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Conformação Proteica em alfa-Hélice , Nucleotídeos Cíclicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismoRESUMO
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.
Assuntos
Canais Iônicos/metabolismo , Animais , AMP Cíclico/farmacologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/química , Ligantes , Miócitos Cardíacos , Neurônios , Técnicas de Patch-Clamp , Canais de Potássio , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Subunidades ProteicasRESUMO
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are voltage-gated tetrameric cation channels that generate electrical rhythmicity in neurons and cardiomyocytes. Activation can be enhanced by the binding of adenosine-3',5'-cyclic monophosphate (cAMP) to an intracellular cyclic nucleotide binding domain. Based on previously determined rate constants for a complex Markovian model describing the gating of homotetrameric HCN2 channels, we analyzed probability fluxes within this model, including unidirectional probability fluxes and the probability flux along transition paths. The time-dependent probability fluxes quantify the contributions of all 13 transitions of the model to channel activation. The binding of the first, third and fourth ligand evoked robust channel opening whereas the binding of the second ligand obstructed channel opening similar to the empty channel. Analysis of the net probability fluxes in terms of the transition path theory revealed pronounced hysteresis for channel activation and deactivation. These results provide quantitative insight into the complex interaction of the four structurally equal subunits, leading to non-equality in their function.