RESUMEN
Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with ß-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and ß-subunits on NaV channels raises the question of whether PRRT2 and ß-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that ß-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the ß4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that ß4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.
Asunto(s)
Proteínas de la Membrana , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso , Neuronas , Humanos , Ataxia , Encéfalo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Neuronas/química , Neuronas/citologíaRESUMEN
Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM.
Asunto(s)
Calmodulina/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Trastorno Autístico/genética , Calmodulina/química , Humanos , Simulación del Acoplamiento Molecular , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/genética , Unión ProteicaRESUMEN
The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.
Asunto(s)
Guanidina/química , Canal de Sodio Activado por Voltaje NAV1.7/química , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Animales , Descubrimiento de Drogas , Ganglios Espinales/metabolismo , Humanos , Canal de Sodio Activado por Voltaje NAV1.1/química , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.3/química , Canal de Sodio Activado por Voltaje NAV1.4/química , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.6/química , Canal de Sodio Activado por Voltaje NAV1.8/química , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: To investigate the relationships among phenotypes, genotypes, and funotypes of SCN2A-related developmental epileptic encephalopathy (DEE). METHODS: We enrolled five DEE patients with five de novo variants of the SCN2A. Functional analysis and pharmacological features of Nav1.2 channel protein expressed in HEK293T cells were characterized by whole-cell patch-clamp recording. RESULTS: The phenotypes of c.4712T>C(p. I1571T), c.2995G>A(p.E999K), and c.4015A>G(p. N1339D) variants showed similar characteristics, including early seizure onset with severe to profound intellectual disability. Electrophysiological recordings revealed a hyperpolarizing shift in the voltage dependence of the activation curve and smaller recovery time constants of fast-inactivation than in wild type, indicating a prominent gain of function (GOF). Moreover, pharmacological electrophysiology showed that phenytoin inhibited over a 70% peak current and was more effective than oxcarbazepine and carbamazepine. In contrast, c.4972C>T (p.P1658S) and c.5317G>A (p.A1773T) led to loss of function (LOF) changes, showing reduced current density and enhanced fast inactivation. Both showed seizure onset after 3 months of age with moderate development delay. Interestingly, we discovered that choreoathetosis was a specific phenotype feature. CONCLUSION: These findings provided the insights into the phenotype-genotype-funotype relationships of SCN2A-related DEE. The preliminary evaluation using the distinct hints of GOF and LOF helped plan the treatment, and the next precise step should be electrophysiological study.
Asunto(s)
Discapacidades del Desarrollo/genética , Epilepsia/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Fenotipo , Potenciales de Acción/efectos de los fármacos , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Preescolar , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/tratamiento farmacológico , Epilepsia/diagnóstico , Epilepsia/tratamiento farmacológico , Femenino , Mutación con Ganancia de Función , Células HEK293 , Humanos , Lactante , Activación del Canal Iónico , Mutación con Pérdida de Función , Masculino , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Medicina de Precisión , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores de los Canales de Sodio/uso terapéuticoRESUMEN
The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo-electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the µ-conotoxin KIIIA, in the presence of an auxiliary subunit, ß2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of ß2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.
Asunto(s)
Conotoxinas/química , Canal de Sodio Activado por Voltaje NAV1.2/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Células HEK293 , Humanos , Conformación Proteica , Subunidad beta-2 de Canal de Sodio Activado por Voltaje/químicaRESUMEN
Although the clinical use of targeted gene sequencing-based diagnostics is valuable, whole-exome sequencing has also emerged as a successful diagnostic tool in molecular genetics laboratories worldwide. Molecular genetic tests for episodic ataxia type 2 (EA2) usually target only the specific calcium channel gene (CACNA1A) that is known to cause EA2. In cases where no mutations are identified in the CACNA1A gene, it is important to identify the causal gene so that more effective treatment can be prioritized for patients. Here we present a case of a proband with a complex episodic ataxias (EA)/seizure phenotype with an EA-affected father; and an unaffected mother, all negative for CACNA1A gene mutations. The trio was studied by whole-exome sequencing to identify candidate genes responsible for causing the complex EA/seizure phenotype. Three rare or novel variants in Sodium channel α2-subunit; SCN2A (c.3973G>T: p.Val1325Phe), Potassium channel, Kv3.2; KCNC2 (c.1006T>C: p.Ser336Pro) and Sodium channel Nav1.6; SCN8A (c.3421C>A: p.Pro1141Thr) genes were found in the proband. While the SCN2A variant is likely to be causal for episodic ataxia, each variant may potentially contribute to the phenotypes observed in this family. This study highlights that a major challenge of using whole-exome/genome sequencing is the identification of the unique causative mutation that is associated with complex disease.
Asunto(s)
Ataxia/diagnóstico , Ataxia/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Fenotipo , Adolescente , Adulto , Canales de Calcio/genética , Análisis Mutacional de ADN , Humanos , Masculino , Modelos Moleculares , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/química , Linaje , Conformación Proteica , Evaluación de Síntomas , Tomografía Computarizada por Rayos X , Secuenciación del Exoma , Adulto JovenRESUMEN
Bacterial and human voltage-gated sodium channels (Navs) exhibit similar cation selectivity, despite their distinct EEEE and DEKA selectivity filter signature sequences. Recent high-resolution structures for bacterial Navs have allowed us to learn about ion conduction mechanisms in these simpler homo-tetrameric channels, but our understanding of the function of their mammalian counterparts remains limited. To probe these conduction mechanisms, a model of the human Nav1.2 channel has been constructed by grafting residues of its selectivity filter and external vestibular region onto the bacterial NavRh channel with atomic-resolution structure. Multi-µs fully atomistic simulations capture long time-scale ion and protein movements associated with the permeation of Na+ and K+ ions, and their differences. We observe a Na+ ion knock-on conduction mechanism facilitated by low energy multi-carboxylate/multi-Na+ complexes, akin to the bacterial channels. These complexes involve both the DEKA and vestibular EEDD rings, acting to draw multiple Na+ into the selectivity filter and promote permeation. When the DEKA ring lysine is protonated, we observe that its ammonium group is actively participating in Na+ permeation, presuming the role of another ion. It participates in the formation of a stable complex involving carboxylates that collectively bind both Na+ and the Lys ammonium group in a high-field strength site, permitting pass-by translocation of Na+. In contrast, multiple K+ ion complexes with the DEKA and EEDD rings are disfavored by up to 8.3 kcal/mol, with the K+-lysine-carboxylate complex non-existent. As a result, lysine acts as an electrostatic plug that partially blocks the flow of K+ ions, which must instead wait for isomerization of lysine downward to clear the path for K+ passage. These distinct mechanisms give us insight into the nature of ion conduction and selectivity in human Nav channels, while uncovering high field strength carboxylate binding complexes that define the more general phenomenon of Na+-selective ion transport in nature.
Asunto(s)
Ácidos Carboxílicos/química , Iones , Lisina/química , Canal de Sodio Activado por Voltaje NAV1.2/química , Aminas/química , Proteínas Bacterianas/química , Humanos , Potenciales de la Membrana , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genética , Potasio/química , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Sodio/química , Electricidad Estática , TermodinámicaRESUMEN
The purpose of this investigation is to contribute to the development of new anticonvulsant drugs to treat patients with refractory epilepsy. We applied a virtual screening protocol that involved the search into molecular databases of new compounds and known drugs to find small molecules that interact with the open conformation of the Nav1.2 pore. As the 3D structure of human Nav1.2 is not available, we first assembled 3D models of the target, in closed and open conformations. After the virtual screening, the resulting candidates were submitted to a second virtual filter, to find compounds with better chances of being effective for the treatment of P-glycoprotein (P-gp) mediated resistant epilepsy. Again, we built a model of the 3D structure of human P-gp, and we validated the docking methodology selected to propose the best candidates, which were experimentally tested on Nav1.2 channels by patch clamp techniques and in vivo by the maximal electroshock seizure (MES) test. Patch clamp studies allowed us to corroborate that our candidates, drugs used for the treatment of other pathologies like Ciprofloxacin, Losartan, and Valsartan, exhibit inhibitory effects on Nav1.2 channel activity. Additionally, a compound synthesized in our lab, N, N'-diphenethylsulfamide, interacts with the target and also triggers significant Na1.2 channel inhibitory action. Finally, in vivo studies confirmed the anticonvulsant action of Valsartan, Ciprofloxacin, and N, N'-diphenethylsulfamide.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Anticonvulsivantes/química , Epilepsia/tratamiento farmacológico , Canal de Sodio Activado por Voltaje NAV1.2/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Anticonvulsivantes/farmacología , Ciprofloxacina/química , Ciprofloxacina/farmacología , Bases de Datos de Compuestos Químicos , Células HEK293 , Humanos , Losartán/química , Losartán/farmacología , Masculino , Ratones , Conformación Molecular , Simulación del Acoplamiento Molecular , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Unión Proteica , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Valsartán/química , Valsartán/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
Nav 1.2, a member of voltage-gated sodium channels (Nav s) that are responsible for the generation and propagation of action potentials along the cell membrane, and play a vital role in the process of information transmission within the nervous system and muscle contraction, is preferentially expressed in the central nervous system. As a potent and selective blocker of Nav s, tetrodotoxin (TTX) has been extensively studied in biological and chemical sciences, whereas the detailed mechanism by which it blocks nine Nav 1 channel subtypes remain elusive. Despite the high structural similarity, the TTX metabolite 4,9-anhydro-TTX is 161 times less effective toward the mammalian Nav 1.2, which puzzled us to ask a question why such a subtle structural variation results in the largely binding affinity difference. In the current work, an integrated computational strategy, including homology modeling, induced fit docking, explicit-solvent MD simulations, and free energy calculations, was employed to investigate the binding mechanism and conformational determinants of TTX analogs. Based on the computational results, the H-bond interactions between C4-OH and C9-OH of TTX and the outer ring carboxylates of the selectivity-filter residues, and the cation-π interaction between the primary amine of guanidinium of TTX and Phe385 determine the difference of their binding affinities. Moreover, the computationally simulations were carried out for the D384N and E945K mutants of hNav 1.2-TTX, and the rank of the predicted binding free energies is in accordance with the experimental data. These observations provide a valuable model to design potent and selective neurotoxins of Nav 1.2 and shed light on the blocking mechanism of TTX to sodium channels.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Tetrodotoxina/metabolismo , Sitios de Unión , Humanos , Enlace de Hidrógeno , Mutagénesis Sitio-Dirigida , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/genética , Unión Proteica , Tetrodotoxina/análogos & derivados , TermodinámicaRESUMEN
Scorpion α-toxins are polypeptides that inhibit voltage-gated sodium channel inactivation. They are divided into mammal, insect and α-like toxins based on their relative activity toward different phyla. Several factors are currently known to influence the selectivity, which are not just particular amino acid residues but also general physical, chemical, and topological properties of toxin structural modules. The objective of this study was to change the selectivity profile of a chosen broadly active α-like toxin, BeM9 from Mesobuthus eupeus, toward mammal-selective. Based on the available information on what determines scorpion α-toxin selectivity, we designed and produced msBeM9, a BeM9 derivative, which was verified to be exclusively active toward mammalian sodium channels and, most importantly, toward the Nav 1.2 isoform expressed in the brain.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.2/química , Neurotoxinas/química , Oocitos/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Insectos/efectos de los fármacos , Insectos/metabolismo , Ratones , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Neurotoxinas/biosíntesis , Neurotoxinas/genética , Neurotoxinas/toxicidad , Oocitos/citología , Oocitos/metabolismo , Unión Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Escorpiones/química , Escorpiones/patogenicidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Tiorredoxinas/biosíntesis , Tiorredoxinas/química , Tiorredoxinas/genética , Xenopus laevisRESUMEN
Human voltage-gated sodium channel NaV1.2 has a single pore-forming α-subunit and two transmembrane ß-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N- CaM (CaMC) bound to an unlabeled peptide with the sequence of rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the NaV1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human NaV1.2 IQ motif (NaV1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with NaV1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in NaV1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to NaV1.2IQp.
Asunto(s)
Apoproteínas/química , Apoproteínas/metabolismo , Calmodulina/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Resonancia Magnética Nuclear Biomolecular , Secuencias de Aminoácidos , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca2+)4-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2IQp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.
Asunto(s)
Secuencias de Aminoácidos , Calcio/farmacología , Calmodulina/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/química , Animales , Sitios de Unión , Calcio/metabolismo , Proteínas del Tejido Nervioso/química , Resonancia Magnética Nuclear Biomolecular , RatasRESUMEN
Improper function of voltage-gated sodium channels (NaVs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target NaVs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of NaV subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of NaV through a systematic structure-activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human NaV1.7 (hNaV1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective NaV inhibitors.
Asunto(s)
Proteínas Musculares/química , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.7/química , Proteínas Recombinantes/química , Saxitoxina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/química , Animales , Sitios de Unión , Células CHO , Cricetulus , Diseño de Fármacos , Expresión Génica , Células HEK293 , Humanos , Cinética , Simulación del Acoplamiento Molecular , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Técnicas de Placa-Clamp , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saxitoxina/química , Bloqueadores de los Canales de Sodio/química , Canales de Sodio/genética , Canales de Sodio/metabolismo , Relación Estructura-Actividad , Tetrodotoxina/química , Tetrodotoxina/farmacologíaRESUMEN
To investigate the mechanisms by which ß-subunits influence Nav channel function, we solved the crystal structure of the ß2 extracellular domain at 1.35Å. We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in ß2 with Nav1.2. We identified a flexible loop formed by (72)Cys and (75)Cys, a unique feature among the four ß-subunit isoforms. Moreover, we found that (55)Cys helps to determine the influence of ß2 on Nav1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that (55)Cys forms a disulfide bond with (910)Cys in the Nav1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Nav1.2 (912/918)Cys residues. The concepts emerging from this work will help to form a model reflecting the ß-subunit location in a Nav channel complex.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Unión Proteica , Conformación Proteica , Mapeo de Interacción de ProteínasRESUMEN
Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.
Asunto(s)
Conotoxinas/química , Disulfuros/química , Proteínas Musculares/química , Canal de Sodio Activado por Voltaje NAV1.2/química , Bloqueadores de los Canales de Sodio/química , Canales de Sodio/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Conotoxinas/síntesis química , Cristalografía por Rayos X , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Caracoles/química , Bloqueadores de los Canales de Sodio/síntesis química , Canales de Sodio/genética , Canales de Sodio/metabolismo , Técnicas de Síntesis en Fase Sólida , Relación Estructura-ActividadRESUMEN
µO§-Conotoxin GVIIJ is a 35-amino acid peptide that readily blocks six of eight tested NaV1 subunit isoforms of voltage-gated sodium channels. µO§-GVIIJ is unusual in having an S-cysteinylated cysteine (at residue 24). A proposed reaction scheme involves the peptide-channel complex stabilized by a disulfide bond formed via thiol-disulfide exchange between Cys24 of the peptide and a Cys residue at neurotoxin receptor site 8 in the pore module of the channel (specifically, Cys910 of rat NaV1.2). To examine this model, we synthesized seven derivatives of µO§-GVIIJ in which Cys24 was disulfide-bonded to various thiols (or SR groups) and tested them on voltage-clamped Xenopus laevis oocytes expressing NaV1.2. In the proposed model, the SR moiety is a leaving group that is no longer present in the final peptide-channel complex; thus, the same koff value should be obtained regardless of the SR group. We observed that all seven derivatives, whose kon values varied over a 30-fold range, had the same koff value. Concordant results were observed with NaV1.6, for which the koff was 17-fold larger. Additionally, we tested two µO§-GVIIJ derivatives (where SR was glutathione or a free thiol) on two NaV1.2 Cys replacement mutants (NaV1.2[C912A] and NaV1.2[C918A]) without and with reduction of channel disulfides by dithiothreitol. The results indicate that Cys910 in wild-type NaV1.2 has a free thiol and conversely suggest that in NaV1.2[C912A] and NaV1.2[C918A], Cys910 is disulfide-bonded to Cys918 and Cys912, respectively. Redox states of extracellular cysteines of sodium channels have hitherto received scant attention, and further experiments with GVIIJ may help fill this void.
Asunto(s)
Conotoxinas/química , Cisteína/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/química , Animales , Sitios de Unión , Conotoxinas/metabolismo , Cisteína/química , Cisteína/genética , Disulfuros/química , Disulfuros/metabolismo , Cinética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Oocitos , Oxidación-Reducción , Ratas , Xenopus laevisRESUMEN
With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3>hNaV1.2>hNaV1.1>hNaV1.6>hNaV1.3>hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel.
Asunto(s)
Benchmarking/métodos , Detergentes/química , Proteínas de la Membrana/química , Canales de Sodio Activados por Voltaje/química , Animales , Células CHO , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Cristalografía por Rayos X , Anguilas/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.1/química , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.3/química , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/ultraestructura , Estabilidad Proteica/efectos de los fármacos , Ratas , Solubilidad , Temperatura , Toxinas Biológicas/metabolismo , Toxinas Biológicas/farmacología , Canales de Sodio Activados por Voltaje/metabolismo , Canales de Sodio Activados por Voltaje/ultraestructuraRESUMEN
Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.
Asunto(s)
Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Neuronas/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/farmacología , Membrana Celular , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Células HEK293 , Humanos , Inmunoprecipitación , Anotación de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/genética , Plasticidad Neuronal , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Unión Proteica , Proteoma/genética , Proteoma/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Phosphorylation plays an essential role in regulating voltage-gated sodium (Na(v)) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Na(v) channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Na(v) channels. METHODS: We used patch-clamp electrophysiology to record sodium currents from Na(v)1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. RESULTS: We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Na(v)1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3ß suppressed Na(v)1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Na(v)1.2 channel indicates that cell surface expression of CD4-Na(v)1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3ß phosphorylates T(1966) at the C-terminal tail of Na(v)1.2. CONCLUSION: These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail. GENERAL SIGNIFICANCE: These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.
Asunto(s)
Glucógeno Sintasa Quinasa 3/fisiología , Canal de Sodio Activado por Voltaje NAV1.2/fisiología , Secuencia de Aminoácidos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células HEK293 , Humanos , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.2/química , FosforilaciónRESUMEN
Animal toxins that inhibit voltage-gated sodium (Na(v)) channel fast inactivation can do so through an interaction with the S3b-S4 helix-turn-helix region, or paddle motif, located in the domain IV voltage sensor. Here, we used surface plasmon resonance (SPR), an optical approach that uses polarized light to measure the refractive index near a sensor surface to which a molecule of interest is attached, to analyze interactions between the isolated domain IV paddle and Na(v) channel-selective α-scorpion toxins. Our SPR analyses showed that the domain IV paddle can be removed from the Na(v) channel and immobilized on sensor chips, and suggest that the isolated motif remains susceptible to animal toxins that target the domain IV voltage sensor. As such, our results uncover the inherent pharmacological sensitivities of the isolated domain IV paddle motif, which may be exploited to develop a label-free SPR approach for discovering ligands that target this region.