RESUMO
The R-type voltage-gated calcium channel CaV2.3 is predominantly located in the presynapse and is implicated in distinct types of epileptic seizures. It has consequently emerged as a molecular target in seizure treatment. Here, we determined the cryo-EM structure of the CaV2.3-α2δ1-ß1 complex in the topiramate-bound state at a 3.0 Å resolution. We provide a snapshot of the binding site of topiramate, a widely prescribed antiepileptic drug, on a voltage-gated ion channel. The binding site is located at an intracellular juxtamembrane hydrophilic cavity. Further structural analysis revealed that topiramate may allosterically facilitate channel inactivation. These findings provide fundamental insights into the mechanism underlying the inhibitory effect of topiramate on CaV and NaV channels, elucidating a previously unseen modulator binding site and thus pointing toward a route for the development of new drugs.
Assuntos
Anticonvulsivantes , Canais de Cálcio Tipo R , Microscopia Crioeletrônica , Topiramato , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Topiramato/química , Topiramato/farmacologia , Humanos , Regulação Alostérica/efeitos dos fármacos , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/metabolismo , Sítios de Ligação , Modelos Moleculares , Células HEK293 , Conformação Proteica , Frutose/química , Frutose/análogos & derivados , Frutose/metabolismo , Animais , Proteínas de Transporte de CátionsRESUMO
Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVß subunits. CaVß subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroß) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVß. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVß, Sroß prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroß and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroß and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroß, similar to the locus found in mammalian CaVß, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVß through a minimally conserved molecular interface.
Assuntos
Canais de Cálcio Tipo R/química , Proteínas de Transporte de Cátions/química , Guanilato Quinases/química , Proteínas de Protozoários/química , Substituição de Aminoácidos , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
During the recording of whole cell currents from stably transfected HEK-293 cells, the decline of currents carried by the recombinant human Cav2.3+ß3 channel subunits is related to adenosine triphosphate (ATP) depletion after rupture of the cells. It reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials (Neumaier F., et al., 2018). Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. These findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. Protein phosphorylation is required for Cav2.3 channel function and could directly influence the normal features of current carried by these channels. Therefore, results from in vitro and in vivo phosphorylation of Cav2.3 are summarized to come closer to a functional analysis of structural variations in Cav2.3 splice variants.
Assuntos
Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Humanos , FosforilaçãoRESUMO
The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium. Furthermore, the L-type calcium channel blocker nifedipine did not prevent cytosolic ET-1 from inducing an increase in nuclear calcium, as opposed to the dual L- and R-type calcium channel blocker isradipine (PN200-110) (in the presence of nifedipine). In conclusion, the preventative effect with PTX and CTX, and the absence of an effect with nifedipine, as well as the blockade by isradipine on cytosolic ET-1-induced increase in nuclear calcium, suggest that this nuclear calcium influx in hVSMCs is due to activation of the steady-state R-type calcium channel. The sarcolemmal and nuclear membrane R-type calcium channels in hVSMCs are involved in ET-1 modulation of vascular tone in physiology and pathology.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Sinalização do Cálcio , Proteínas de Transporte de Cátions/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Endotelina-1/metabolismo , Músculo Liso Vascular/metabolismo , Membrana Nuclear/metabolismo , Aorta , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo R/química , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte de Cátions/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Toxina da Cólera/farmacologia , Citosol/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Imageamento Tridimensional , Fusão de Membrana/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Microscopia Confocal , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Membrana Nuclear/efeitos dos fármacos , Toxina Pertussis/farmacologia , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismoRESUMO
We present a simplified reaction network in a single well-mixed volume that captures the general features of CaMKII dynamics observed during both synaptic input and spine depolarization. Our model can also account for the greater-than-control CaMKII activation observed with added EGTA during depolarization. Calcium input currents are modeled after experimental observations, and existing models of calmodulin and CaMKII autophosphorylation are used. After calibration against CaMKII activation data in the absence of chelators, CaMKII activation dynamics due to synaptic input via n-methyl-d-aspartate receptors are qualitatively accounted for in the presence of the chelators EGTA and BAPTA without additional adjustments to the model. To account for CaMKII activation dynamics during spine depolarization with added EGTA or BAPTA, the model invokes the modulation of CaV2.3 (R-type) voltage-dependent calcium channel (VDCC) currents observed in the presence of EGTA or BAPTA. To our knowledge, this is a novel explanation for the increased CaMKII activation seen in dendritic spines with added EGTA, and suggests that differential modulation of VDCCs by EGTA and BAPTA offers an alternative or complementary explanation for other experimental results in which addition of EGTA or BAPTA produces different effects. Our results also show that a simplified reaction network in a single, well-mixed compartment is sufficient to account for the general features of observed CaMKII dynamics.
Assuntos
Quelantes de Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dendritos/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Animais , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/metabolismo , Quelantes de Cálcio/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Dendritos/efeitos dos fármacos , Ácido Egtázico/química , Modelos BiológicosRESUMO
Voltage-gated Ca(2+) channels (VGCCs) are ubiquitous in excitable cells. These channels play key roles in many physiological events like cardiac regulation/pacemaker activity due to intracellular Ca(2+) transients. In the myocardium, the Cav1 subfamily (L-type: Cav1.2 and Cav1.3) is the main contributor to excitation-contraction coupling and/or pacemaking, whereas the Cav3 subfamily (T-type: Cav3.1 and Cav3.2) is important in rhythmically firing of the cardiac nodal cells. No established cardiac function has been attributed to the Cav2 family (E-/R-type: Cav2.3) despite accumulating evidence of cardiac dysregulation observed upon deletion of the Cav2.3 gene, the only member of this family so far detected in cardiomyocytes. In this review, we summarize the pathophysiological changes observed after ablation of the E-/R-type VGCC and propose a cardiac mechanism of action for this channel. Also, considering the role played by this channel in epilepsy and its reported sensitivity to antiepileptic drugs, a putative involvement of this channel in the cardiac mechanism of sudden unexpected death in epilepsy is also discussed.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo R/fisiologia , Canais de Cálcio Tipo T/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Morte Súbita/etiologia , Epilepsia/fisiopatologia , Coração/fisiologia , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo T/química , Proteínas de Transporte de Cátions/química , Epilepsia/complicações , HumanosRESUMO
R-type calcium channels (RTCCs) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, we characterized the regional and subcellular localization of Ca(v)2.3 in mice and rats at both light and electron microscopic levels. Ca(v)2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique, or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained <20 immunogold particles, with an average density of â¼260 immunoparticles per µm(3) spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Ca(v)2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Canais de Cálcio Tipo R/ultraestrutura , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Análise de Variância , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/deficiência , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/deficiência , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Epitopos/metabolismo , Feminino , Cobaias , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Imunoeletrônica , Peptídeos/metabolismo , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Ratos , Estatística como Assunto , Estatísticas não Paramétricas , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.
Assuntos
Processamento Alternativo , Canais de Cálcio Tipo R/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Calmodulina/química , Calmodulina/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Células HEK293 , Humanos , Imunoprecipitação , Dados de Sequência MolecularRESUMO
Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Ca(v)2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Ca(v)2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1-IS2 (H111) and IS2-IS3 (H179 and H183) loops potentiates Ca(v)2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Ca(v)2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the "ON" position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Ca(v)2.3, potentially affecting synaptic transmission and plasticity in the brain.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Biofísicos , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/agonistas , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cobre/farmacologia , Ácido Glutâmico/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Células HEK293 , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Ratos Transgênicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oligoelementos/farmacologiaRESUMO
Maintaining blood glucose homeostasis is a complex process that depends on pancreatic islet hormone secretion. Hormone secretion from islets is coupled to calcium entry which results from regenerative islet cell electrical activity. Therefore, the ionic mechanisms that regulate calcium entry into islet cells are crucial for maintaining normal glucose homeostasis. Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs), including five located in or near ion-channel or associated subunit genes, which show an association with human diseases characterized by dysglycemia. This review focuses on polymorphisms and mutations in ion-channel genes that are associated with perturbations in human glucose homeostasis and discusses their potential roles in modulating pancreatic islet hormone secretion.
Assuntos
Glicemia/metabolismo , Homeostase , Insulina/metabolismo , Canais Iônicos/genética , Ilhotas Pancreáticas/metabolismo , Animais , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Transtornos do Metabolismo de Glucose/sangue , Transtornos do Metabolismo de Glucose/genética , Transtornos do Metabolismo de Glucose/metabolismo , Humanos , Secreção de Insulina , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais KATP/química , Canais KATP/genética , Canais KATP/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Mutação , Polimorfismo de Nucleotídeo Único , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismoRESUMO
BACKGROUND: Voltage gated calcium channels (VGCCs) regulate cellular activity in response to membrane depolarization by altering calcium homeostasis. Because calcium is the most versatile second messenger, regulation of the amount of VGCCs at the plasma membrane is highly critical for several essential cellular processes. Among the different types of VGCCs, the Ca(v)2.3 calcium channel and its regulation mechanisms are least understood due to Ca(v)2.3's resistance to most pharmacological agents. METHODS: In order to study regulation and surface expression of Ca(v)2.3, a yeast two hybrid (Y2H) screen with the II-III loop of human Ca(v)2.3 as bait, was performed. APLP1, a member of the APP gene family and Rab5A, an endocytotic catalyst were identified as putative interaction partners. The interaction were confirmed by immunoprecipitation. To study the functional importance of the interaction, patch-clamp recordings in Ca(v)2.3 stably transfected HEK-293 cells (2C6) and surface biotin endocytosis assays were performed. RESULTS: We are able to show that the II-III loop of the Ca(v)2.3 calcium channel binds APLP1 and that this binding promotes internalization of the channel. In addition, Rab5A also binds to the same loop of the channel and exerts an inhibitory effect on APLP1 mediated channel internalization. CONCLUSIONS: This study identifies a regulation mechanism of Ca(v)2.3's surface expression, which implicates APLP1 as a regulator of calcium homeostasis. Thus APLP1 may play a crucial role in neuropathological mechanisms, which involve modulation of surface expression of voltage-gated Ca(2+) channels.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Precursor de Proteína beta-Amiloide/genética , Biotina/genética , Biotina/metabolismo , Canais de Cálcio Tipo R/química , Proteínas de Transporte de Cátions/química , Endocitose , Células HEK293 , Humanos , Imunoprecipitação , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Transfecção , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Mutations in distal S6 were shown to significantly alter the stability of the open state of Ca(V)2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944-27952). By analogy with K(V) channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca(V)2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20 mV shift in its charge-voltage relationship as compared with Ca(V)2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG(interact) > 2 kcal mol(-1)) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG(interact) ≈6 kcal mol(-1) suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca(V) channel.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Leucina/metabolismo , Mutação Puntual , Sequência de Aminoácidos , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , DNA Recombinante/genética , Humanos , Dados de Sequência Molecular , Técnicas de Patch-ClampRESUMO
BACKGROUND: Calcium channels are essential in coupling action potential to signal transduction in cells. There are several types of calcium channels, which can be pharmacologically classified as L-, N-, P/Q-, R- and T-type. But molecular basis of R-type channels is less clearly understood compared the other channel types. Therefore the current study aims at understanding the molecular functions of R-type calcium channels by identifying interaction partners of the channel. METHODS: In order to do so, a yeast two hybrid (Y2H) screen, with carboxy terminus of α1 subunit of the channel, as the bait, was performed. G1 subunit of v-ATPase was identified as a putative interaction partner of human Ca(v)2.3 by using the Y2H screening. The interaction was confirmed by immunoprecipitation. To study the functional importance of the interaction, bafilomycin A(1), a potent and specific inhibitor of v-ATPase was used in patch-clamp recordings in Ca(v)2.3 stably-transfected HEK-293 cells (2C6) as well as in electroretinography of the isolated bovine retina expressing R-type Ca(2+) channels. RESULTS: G1 subunit of v-ATPase interacts with C-terminal tail of Ca(v)2.3 and bafilomycin A(1) reduces Ca(v)2.3 mediated calcium currents. Additionally peak I(Ca) is inhibited in retinal signal transduction when recorded as ERG b-wave. CONCLUSIONS: The results suggest that v-ATPase interacts physically and also functionally with Ca(v)2.3. This is the first demonstration of association of Ca(v)2.3 C-terminus with a protein complex which is involved in transmembrane signalling.
Assuntos
Adenosina Trifosfatases/metabolismo , Canais de Cálcio Tipo R/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Retina/fisiologia , Transdução de Sinais/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Animais , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Bovinos , Eletrorretinografia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Imunoprecipitação , Ativação do Canal Iônico/efeitos dos fármacos , Macrolídeos/farmacologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Plasmídeos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Extracellular acidification decreases Ca(2+) current amplitude and produces a depolarizing shift in the activation potential (Va) of voltage-gated Ca(2+) channels (VGCC). These effects are common to all VGCC, but differences exist between Ca(2+) channel types and the underlying molecular mechanisms remain largely unknown. We report here that the changes in current amplitude induced by extracellular acidification or alkalinisation are more important for Cav2.3 R type than for Cav2.1 P/Q-type Ca(2+) channels. This difference results from a higher shift of Va combined with a modification of channel conductance. Although involved in the sensitivity of channel conductance to extracellular protons, neither the EEEE locus nor the divalent cation selectivity locus could explain the specificity of the pH effects. We show that this specificity involves two separate sets of amino acids within domain I of the Cavα subunit. Residues of the voltage sensor domain and residues in the pore domain mediate the effects of extracellular protons on Va and on channel conductance, respectively. These new insights are important for elucidating the molecular mechanisms that control VGCC gating and conductance and for understanding the role of extracellular protons in other channels or membrane-tethered enzymes with similar pore and/or voltage sensor domains.
Assuntos
Aminoácidos/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Ativação do Canal Iônico/fisiologia , Prótons , Sequência de Aminoácidos , Aminoácidos/química , Animais , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Alinhamento de Sequência , XenopusRESUMO
UNLABELLED: Cell swelling-induced insulin secretion represents an alternative pathway of stimulation of insulin secretion. INS-1E rat tumor beta cells do not release insulin in response to cell swelling in presence of Ca(2+) despite a good response to glucose challenge and appropriate increase in cell volume. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity. Objective of this study was further characterization of the role of Ca(2+) in secretory process in INS-1 and INS-1E cell lines. Ca(2+) depleted hypotonic medium with 10 muM BAPTA/AM (intracellular chelator) induced insulin secretion from both types of cells. We demonstrated expression of L-type Ca(2+) channel Ca(v)1.2 and non-L-type Ca(2+) channels Ca(v)2.1 (P/Q-type), Ca(v)2.2 (N-type), and Ca(v)3.1 (T-type) in both cell lines. Inhibition of L type channel with nifedipine and/or P/Q type with omega-agatoxin IVA enabled distinct response to hypotonic medium also in INS-1E cells. Tetanus toxin (TeTx) in medium containing Ca(2+) and a group of calcium channel blockers inhibited hypotonicity-induced insulin secretion from INS-1 cells but not from INS-1E cells. CONCLUSION: Hypotonicity-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca(2+), does not require intracellular Ca(2+) and is TeTx resistant.
Assuntos
Cálcio/farmacologia , Insulina/metabolismo , Toxina Tetânica/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo P/química , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/química , Canais de Cálcio Tipo Q/genética , Canais de Cálcio Tipo Q/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular Tumoral , Tamanho Celular , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Soluções Hipotônicas/química , Secreção de Insulina , Masculino , Nifedipino/farmacologia , Ratos , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , ômega-Agatoxina IVA/farmacologiaRESUMO
Calcium entry through voltage-gated calcium channels (VGCC) initiates diverse cellular functions. VGCC pore-forming subunit (Ca(V)alpha(1)) contains four homology repeats, each encompassing a voltage sensor and a pore domain. Three main classes of Ca(V)alpha(1) subunits have been described, Ca(V)1, Ca(V)2 and Ca(V)3 that differ in their voltage-dependence of activation and in the extent in which this process is modulated by the auxiliary beta-subunit (Ca(V)beta). Association of Ca(V)beta induces a coil-to-helix conformation of the I-II intracellular linker joining the first and second repeat of Ca(V)alpha(1) that is thought to be crucial for modulation of channel function. When expressed in Xenopus laevis oocytes in the absence of Ca(V)beta, the voltage to reach 50% activation (V(0.5)) for Ca(V)1.2 and Ca(V)2.3 differs by more than 60 mV and the channel current-carrying capacity by more than thirty-fold. Here we report that the difference in V(0.5) is reduced to about 30 mV and the current-carrying capacity becomes virtually identical when the I-II linkers of Ca(V)1.2 and Ca(V)2.3 are swapped. Co-expression with Ca(V)beta increases the current-carrying capacity of chimeric channels by the same extent, while the difference in V(0.5) with respect to their corresponding parental channels vanishes. Our findings indicate that Ca(V)beta modulatory potency is determined by both, the nature of the I-II linker and the pore-forming subunit background. Moreover, they demonstrate that the I-II linker encodes self-reliant molecular determinants for channel activation and suggest that besides the secondary structure adopted by this segment upon Ca(V)beta association, its chemical nature is as well relevant.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo R/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ativação do Canal Iônico , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Potenciais da Membrana , Dados de Sequência Molecular , Oócitos , Conformação Proteica , Estrutura Terciária de Proteína , Coelhos , Ratos , Relação Estrutura-Atividade , Fatores de Tempo , Xenopus laevisRESUMO
Calmodulin (CaM) regulation of Ca(2+) channels is central to Ca(2+) signaling. Ca(V)1 versus Ca(V)2 classes of these channels exhibit divergent forms of regulation, potentially relating to customized CaM/IQ interactions among different channels. Here we report the crystal structures for the Ca(2+)/CaM IQ domains of both Ca(V)2.1 and Ca(V)2.3 channels. These highly similar structures emphasize that major CaM contacts with the IQ domain extend well upstream of traditional consensus residues. Surprisingly, upstream mutations strongly diminished Ca(V)2.1 regulation, whereas downstream perturbations had limited effects. Furthermore, our Ca(V)2 structures closely resemble published Ca(2+)/CaM-Ca(V)1.2 IQ structures, arguing against Ca(V)1/2 regulatory differences based solely on contrasting CaM/IQ conformations. Instead, alanine scanning of the Ca(V)2.1 IQ domain, combined with structure-based molecular simulation of corresponding CaM/IQ binding energy perturbations, suggests that the C lobe of CaM partially dislodges from the IQ element during channel regulation, allowing exposed IQ residues to trigger regulation via isoform-specific interactions with alternative channel regions.
Assuntos
Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo R/química , Cálcio/química , Calmodulina/química , Modelos Moleculares , Alanina/genética , Sequência de Aminoácidos , Canais de Cálcio Tipo N/genética , Cristalografia por Raios X , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
We recently reported that a histidine (H191) in the S3-S4 loop of domain I is critical for nickel inhibition of the Cav3.2 T-type Ca2+ channel. As in Cav3.2, two histidine residues are commonly found in the IS3-IS4 loops of mammalian Cav2.3 Ca2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Cav2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3-IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Cav2.3.
Assuntos
Canais de Cálcio Tipo R/química , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/química , Histidina/metabolismo , Níquel/farmacologia , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , XenopusRESUMO
The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca(V)1 and Ca(V)2 families. Herein we show that glycine substitution of the VAVIM motif in Ca(V)2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G approximately V1720G), similar (V1718G), or faster (I1721G approximately M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a approximately +10 mV shift in its voltage dependence of activation (E(0.5,act)). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of approximately -20 mV in their E(0.5,act) indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val (349) in IS6, Ile(701) in IIS6, and Leu(1420) in IIIS6 at positions predicted to face Val(1720) in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of Ca(V)2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val(1720) in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in Ca(V)2.3.
Assuntos
Canais de Cálcio Tipo R/química , Proteínas de Transporte de Cátions/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Humanos , Cinética , Canal de Potássio Kv1.2/química , Conformação Molecular , Dados de Sequência Molecular , Oócitos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , XenopusRESUMO
The roles of voltage-sensitive sodium (Na) and calcium (Ca) channels located on dendrites and spines in regulating synaptic signals are largely unknown. Here we use 2-photon glutamate uncaging to stimulate individual spines while monitoring uncaging-evoked excitatory postsynaptic potentials (uEPSPs) and Ca transients. We find that, in CA1 pyramidal neurons in acute mouse hippocampal slices, CaV(2.3) voltage-sensitive Ca channels (VSCCs) are found selectively on spines and act locally to dampen uncaging-evoked Ca transients and somatic potentials. These effects are mediated by a regulatory loop that requires opening of CaV(2.3) channels, voltage-gated Na channels, small conductance Ca-activated potassium (SK) channels, and NMDA receptors. Ca influx through CaV(2.3) VSCCs selectively activates SK channels, revealing the presence of functional Ca microdomains within the spine. Our results suggest that synaptic strength can be modulated by mechanisms that regulate voltage-gated conductances within the spine but do not alter the properties or numbers of synaptic glutamate receptors.