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1.
Clin Epigenetics ; 11(1): 102, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300040

RESUMO

BACKGROUND: Apabetalone (RVX-208) is a bromodomain and extraterminal protein inhibitor (BETi) that in phase II trials reduced the relative risk (RR) of major adverse cardiac events (MACE) in patients with cardiovascular disease (CVD) by 44% and in diabetic CVD patients by 57% on top of statins. A phase III trial, BETonMACE, is currently assessing apabetalone's ability to reduce MACE in statin-treated post-acute coronary syndrome type 2 diabetic CVD patients with low high-density lipoprotein C. The leading cause of MACE is atherosclerosis, driven by dysfunctional lipid metabolism and chronic vascular inflammation (VI). In vitro studies have implicated the BET protein BRD4 as an epigenetic driver of inflammation and atherogenesis, suggesting that BETi may be clinically effective in combating VI. Here, we assessed apabetalone's ability to regulate inflammation-driven gene expression and cell adhesion in vitro and investigated the mechanism by which apabetalone suppresses expression. The clinical impact of apabetalone on mediators of VI was assessed with proteomic analysis of phase II CVD patient plasma. RESULTS: In vitro, apabetalone prevented inflammatory (TNFα, LPS, or IL-1ß) induction of key factors that drive endothelial activation, monocyte recruitment, adhesion, and plaque destabilization. BRD4 abundance on inflammatory and adhesion gene promoters and enhancers was reduced by apabetalone. BRD2-4 degradation by MZ-1 also prevented TNFα-induced transcription of monocyte and endothelial cell adhesion molecules and inflammatory mediators, confirming BET-dependent regulation. Transcriptional regulation by apabetalone translated into a reduction in monocyte adhesion to an endothelial monolayer. In a phase II trial, apabetalone treatment reduced the abundance of multiple VI mediators in the plasma of CVD patients (SOMAscan® 1.3 k). These proteins correlate with CVD risk and include adhesion molecules, cytokines, and metalloproteinases. Ingenuity® Pathway Analysis (IPA®) predicted that apabetalone inhibits pro-atherogenic regulators and pathways and prevents disease states arising from leukocyte recruitment. CONCLUSIONS: Apabetalone suppressed gene expression of VI mediators in monocytes and endothelial cells by inhibiting BET-dependent transcription induced by multiple inflammatory stimuli. In CVD patients, apabetalone treatment reduced circulating levels of VI mediators, an outcome conducive with atherosclerotic plaque stabilization and MACE reduction. Inhibition of inflammatory and adhesion molecule gene expression by apabetalone is predicted to contribute to MACE reduction in the phase III BETonMACE trial.

2.
Atherosclerosis ; 280: 75-84, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30476723

RESUMO

BACKGROUND AND AIMS: Apabetalone is an inhibitor of bromodomain and extraterminal (BET) proteins. In clinical trials, apabetalone reduced the incidence of major adverse cardiac events (MACE) in patients with cardiovascular disease and reduced circulating factors that promote vascular calcification (VC). Because VC contributes to MACE, effects of apabetalone on pro-calcific processes were examined. METHODS AND RESULTS: Apabetalone inhibited extracellular calcium deposition and opposed induction of transdifferentiation markers in human coronary artery vascular smooth muscle cells (VSMCs) under osteogenic culture conditions. Tissue-nonspecific alkaline phosphatase (TNAP) is a key contributor to VC, and apabetalone suppressed osteogenic induction of the mRNA, protein and enzyme activity. The liver is a major source of circulating TNAP, and apabetalone also downregulated TNAP expression in primary human hepatocytes. BRD4, a transcriptional regulator and target of apabetalone, has been linked to calcification. Osteogenic transdifferentiation of VSMCs resulted in disassembly of 100 BRD4-rich enhancers, with concomitant enlargement of remaining enhancers. Apabetalone reduced the size of BRD4-rich enhancers, consistent with disrupting BRD4 association with chromatin. 38 genes were uniquely associated with BRD4-rich enhancers in osteogenic conditions; 11 were previously associated with calcification. Apabetalone reduced levels of BRD4 on many of these enhancers, which correlated with decreased expression of the associated gene. Bioinformatics revealed BRD4 may cooperate with 7 specific transcription factors to promote transdifferentiation and calcification. CONCLUSIONS: Apabetalone counters transdifferentiation and calcification of VSMCs via an epigenetic mechanism involving specific transcription factors. The mechanistic findings, combined with evidence from clinical trials, support further development of apabetalone as a therapeutic for VC.

3.
Kidney Int Rep ; 3(3): 711-721, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29854980

RESUMO

Introduction: Apabetalone, a small molecule inhibitor, targets epigenetic readers termed BET proteins that contribute to gene dysregulation in human disorders. Apabetalone has in vitro and in vivo anti-inflammatory and antiatherosclerotic properties. In phase 2 clinical trials, this drug reduced the incidence of major adverse cardiac events in patients with cardiovascular disease. Chronic kidney disease is associated with a progressive loss of renal function and a high risk of cardiovascular disease. We studied the impact of apabetalone on the plasma proteome in patients with impaired kidney function. Methods: Subjects with stage 4 or 5 chronic kidney disease and matched controls received a single dose of apabetalone. Plasma was collected for pharmacokinetic analysis and for proteomics profiling using the SOMAscan 1.3k platform. Proteomics data were analyzed with Ingenuity Pathway Analysis to identify dysregulated pathways in diseased patients, which were targeted by apabetalone. Results: At baseline, 169 plasma proteins (adjusted P value <0.05) were differentially enriched in renally impaired patients versus control subjects, including cystatin C and ß2 microglobulin, which correlate with renal function. Bioinformatics analysis of the plasma proteome revealed a significant activation of 42 pathways that control immunity and inflammation, oxidative stress, endothelial dysfunction, vascular calcification, and coagulation. At 12 hours postdose, apabetalone countered the activation of pathways associated with renal disease and reduced the abundance of disease markers, including interleukin-6, plasminogen activator inhibitor-1, and osteopontin. Conclusion: These data demonstrated plasma proteome dysregulation in renally impaired patients and the beneficial impact of apabetalone on pathways linked to chronic kidney disease and its cardiovascular complications.

4.
Genet Med ; 20(5): 503-512, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28933792

RESUMO

PurposeCaV3.2 signaling contributes to nociception, pruritus, gastrointestinal motility, anxiety, and blood pressure homeostasis. This calcium channel, encoded by CACNA1H, overlaps the human tryptase locus, wherein increased TPSAB1 copy number causes hereditary α-tryptasemia. Germ-line CACNA1H variants may contribute to the variable expressivity observed with this genetic trait.MethodsTryptase-encoding sequences at TPSAB1 and TPSB2, and TPSG1 and CACNA1H variants were genotyped in 46 families with hereditary α-tryptasemia syndrome. Electrophysiology was performed on tsA201 HEK cells transfected with wild-type or variant CACNA1H constructs. Effects on clinical phenotypes were interrogated in families with TPSAB1 duplications and in volunteers from the ClinSeq cohort.ResultsThree nonsynonymous variants in CACNA1H (rs3751664, rs58124832, and rs72552056) cosegregated with TPSAB1 duplications in 32/46 families and were confirmed to be in linkage disequilibrium (LD). In vitro, variant CaV3.2 had functional effects: reducing current densities, and altering inactivation and deactivation properties. No clinical differences were observed in association with the CACNA1H haplotype.ConclusionA previously unrecognized haplotype containing three functional CACNA1H variants is relatively common among Caucasians, and is frequently coinherited on the same allele as additional TPSAB1 copies. The variant CACNA1H haplotype, which in vitro imparts partial gain of function, does not result in detectable phenotypic differences in the heterozygous state.


Assuntos
Canais de Cálcio Tipo T/genética , Variações do Número de Cópias de DNA , Frequência do Gene , Haplótipos , Padrões de Herança , Triptases/genética , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular , Duplicação Gênica , Estudos de Associação Genética , Loci Gênicos , Técnicas de Genotipagem , Humanos , Desequilíbrio de Ligação , Mutação , Fenótipo , Análise de Sequência de DNA , Triptases/metabolismo
5.
Glia ; 63(11): 2023-2039, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26119281

RESUMO

Axonal transsynaptic signaling between presynaptic neurexin (NX) and postsynaptic neuroligin (NL) is essential for many properties of synaptic connectivity. Here, we demonstrate the existence of a parallel axo-glial signaling pathway between axonal NX and oligodendritic (OL) NL3. We show that this pathway contributes to the regulation of myelinogenesis, the maintenance of established myelination, and the differentiation state of the OL using in vitro models. We first confirm that NL3 mRNA and protein are expressed in OLs and in OL precursors. We then show that OLs in culture form contacts with non-neuronal cells exogenously expressing NL3's binding partners NX1α or NX1ß. Conversely, blocking axo-glial NX-NL3 signaling by saturating NX with exogenous soluble NL protein (NL-ECD) disrupts interactions between OLs and axons in both in vitro and ex vivo assays. Myelination by OLs is tied to their differentiation state, and we find that blocking NX-NL signaling with soluble NL protein also caused OL differentiation to stall at an immature stage. Moreover, in vitro knockdown of NL3 in OLs with siRNAs stalls their development and reduces branching complexity. Interestingly, inclusion of an autism related mutation in the NL blocking protein attenuates these effects; OLs differentiate and the dynamics of OL-axon signaling occur normally as this peptide does not disrupt NX-NL3 axo-glial interactions. Our findings provide evidence not only for a new pathway in axo-glial communication, they also potentially explain the correlation between altered white matter and autism. GLIA 2015;63:2023-2039.

6.
Mol Brain ; 7: 42, 2014 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-24886461

RESUMO

BACKGROUND: Inorganic polyphosphate (polyP) is a highly charged polyanion capable of interacting with a number of molecular targets. This signaling molecule is released into the extracellular matrix by central astrocytes and by peripheral platelets during inflammation. While the release of polyP is associated with both induction of blood coagulation and astrocyte extracellular signaling, the role of secreted polyP in regulation of neuronal activity remains undefined. Here we test the hypothesis that polyP is an important participant in neuronal signaling. Specifically, we investigate the ability of neurons to release polyP and to induce neuronal firing, and clarify the underlying molecular mechanisms of this process by studying the action of polyP on voltage gated channels. RESULTS: Using patch clamp techniques, and primary hippocampal and dorsal root ganglion cell cultures, we demonstrate that polyP directly influences neuronal activity, inducing action potential generation in both PNS and CNS neurons. Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels. Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells. Using in situ DAPI localization and live imaging, we demonstrate that polyP is naturally present in synaptic regions and is released from the neurons upon depolarization. Finally, using a biochemical assay we demonstrate that polyP is present in synaptosomes and can be released upon their membrane depolarization by the addition of potassium chloride. CONCLUSIONS: We conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels. Together, our data establishes that polyP could function as excitatory neuromodulator in both the PNS and CNS.


Assuntos
Potenciais de Ação/fisiologia , Ativação do Canal Iônico/fisiologia , Neurônios/fisiologia , Polifosfatos/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Técnicas de Cocultura , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Indóis/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
7.
PLoS One ; 7(10): e46865, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056495

RESUMO

When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Cl(swell)) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Cl(swell) channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila Cl(swell) channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Cl(swell) current (I(Clswell)). We further demonstrate the essential contribution of dBest1 to Drosophila I(Clswell) with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous I(Clswell). We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila-like I(Clswell). In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Cl(swell) channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.


Assuntos
Canais de Cloreto/deficiência , Canais de Cloreto/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genômica , Proteínas Luminescentes/metabolismo , Interferência de RNA , Sequência de Aminoácidos , Animais , Bestrofinas , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Humanos , Camundongos , Dados de Sequência Molecular , Mutação
8.
Channels (Austin) ; 6(3): 157-65, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22677788

RESUMO

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through ß adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/genética , Linhagem Celular Transformada , Deleção de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Oócitos , Técnicas de Patch-Clamp , Prolina/metabolismo , Domínios Proteicos Ricos em Prolina , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Transporte Proteico/genética , Xenopus
9.
Proc Natl Acad Sci U S A ; 108(48): 19234-9, 2011 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-22084111

RESUMO

Specialized proteins in the plasma membrane, endoplasmic reticulum (ER), and mitochondria tightly regulate intracellular calcium. A unique mechanism called store-operated calcium entry is activated when ER calcium is depleted, serving to restore intra-ER calcium levels. An ER calcium sensor, stromal interaction molecule 1 (STIM1), translocates within the ER membrane upon store depletion to the juxtaplasma membrane domain, where it interacts with intracellular domains of a highly calcium-selective plasma membrane ion channel, Orai1. STIM1 gates Orai1, allowing calcium to enter the cytoplasm, where it repletes the ER store via calcium-ATPases pumps. Here, we performed affinity purification of Orai1 from Jurkat cells to identify partner of STIM1 (POST), a 10-transmembrane-spanning segment protein of unknown function. The protein is located in the plasma membrane and ER. POST-Orai1 binding is store depletion-independent. On store depletion, the protein binds STIM1 and moves within the ER to localize near the cell membrane. This protein, TMEM20 (POST), does not affect store-operated calcium entry but does reduce plasma membrane Ca(2+) pump activity. Store depletion promotes STIM1-POST complex binding to smooth ER and plasma membrane Ca(2+) ATPases (SERCAs and PMCAs, respectively), Na/K-ATPase, as well as to the nuclear transporters, importins-ß and exportins.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Biológico/fisiologia , Canais de Cálcio/metabolismo , Linhagem Celular , Eletrofisiologia , Humanos , Microscopia de Fluorescência , Proteína ORAI1 , Proteínas Carreadoras de Solutos , Molécula 1 de Interação Estromal
10.
PLoS One ; 3(5): e2082, 2008 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-18461159

RESUMO

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.


Assuntos
Gânglios Espinais/fisiologia , Monoterpenos/farmacologia , Neurônios/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Cinética , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Estereoisomerismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/efeitos dos fármacos
11.
J Biol Chem ; 281(31): 22085-91, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16754686

RESUMO

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca(V)1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca(V)3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca(V)3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.


Assuntos
Transtorno Autístico/genética , Canais de Cálcio Tipo T/genética , Mutação de Sentido Incorreto , Transtorno Autístico/epidemiologia , Transtorno Autístico/etiologia , Canais de Cálcio Tipo T/metabolismo , Estudos de Casos e Controles , Sequência Conservada , Análise Mutacional de DNA , Eletrofisiologia , Saúde da Família , Humanos , Cinética , Epidemiologia Molecular , Linhagem
12.
J Biol Chem ; 281(20): 14015-25, 2006 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-16554304

RESUMO

L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following alpha5beta1 integrin activation. Only the alpha1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by alpha5beta1 integrin requires phosphorylation of alpha1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following alpha5beta1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Integrina alfa5beta1/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Encéfalo/metabolismo , Dados de Sequência Molecular , Músculos/metabolismo , Miócitos de Músculo Liso/metabolismo , Neurônios/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Suínos , Quinases da Família src
13.
J Physiol ; 554(Pt 2): 263-73, 2004 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12815185

RESUMO

Voltage-dependent inactivation of calcium channels is a key mechanism for regulating intracellular calcium levels and neuronal excitability. In sodium and potassium channels, the molecular determinants that govern fast inactivation involve pore block by a cytoplasmic gating particle. As we discuss here, there is an increasing body of evidence that is consistent with a qualitatively similar inactivation mechanism in high-voltage-activated calcium channels. Work from a number of laboratories has implicated both cytoplasmic regions and the pore-lining S6 transmembrane helices in the inactivation process. Together with our recent findings, this leads us to propose a model in which the intracellular domain I-II linker region acts as a 'hinged lid' that physically occludes the pore by docking to the cytoplasmic ends of the S6 segments. We further propose that the ancillary calcium channel Beta subunits differentially modulate inactivation kinetics by binding to and thereby regulating the mobility of the putative inactivation gate. Indeed, additional evidence suggests that the carboxy terminus, amino terminus and domain III-IV linker regions of the channel modulate inactivation rates through interactions with the I-II linker per se, or indirectly via the ancillary Beta subunits. Taken together, the fast voltage-dependent inactivation of calcium channels appears reminiscent of that of sodium channels, but appears to show a more complex regulation through intramolecular interactions between the putative inactivation gate and other cytoplasmic regions.


Assuntos
Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio/química , Canais de Cálcio/fisiologia , Membrana Celular/fisiologia , Animais , Canais de Cálcio/metabolismo , Membrana Celular/ultraestrutura , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Estrutura Secundária de Proteína
14.
J Biol Chem ; 279(5): 3793-800, 2004 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-14602720

RESUMO

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.


Assuntos
Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Linhagem Celular , Eletrofisiologia , Deleção de Genes , Humanos , Cinética , Dados de Sequência Molecular , Fenótipo , Testes de Precipitina , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Transfecção
15.
J Med Chem ; 46(1): 87-96, 2003 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-12502362

RESUMO

A series of 4-isoxazolyl-1,4-dihydropyridines (IDs) were prepared and characterized, and their interaction with the calcium channel was studied by patch clamp analysis. The structure-activity relationship (SAR) that emerges is distinct from the 4-aryldihydropyridines (DHPs), and affinity increases dramatically at higher holding potentials. Thus, among the 3'-arylisoxazolyl analogues p-Br > p-Cl >> p-F, and p-Cl > m-Cl > o-Cl >> o-MeO. Four of the analogues were examined by single-crystal X-ray diffractometry, and all were found to adopt an O-exo conformation in the solid state. The calculated barrier to rotation, however, suggests that rotation about the juncture between the heterocyclic rings is plausible under physiological conditions. A variable-temperature NMR study confirmed the computation. With Striessnig's computational sequence homologation procedure, a working hypothesis was derived from the data that explains the unique SAR for IDs.


Assuntos
Canais de Cálcio/efeitos dos fármacos , Di-Hidropiridinas/síntese química , Isoxazóis/síntese química , Canais de Cálcio/fisiologia , Linhagem Celular , Cristalografia por Raios X , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Humanos , Isoxazóis/química , Isoxazóis/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Técnicas de Patch-Clamp , Relação Estrutura-Atividade
16.
J Biol Chem ; 277(37): 33598-603, 2002 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-12114507

RESUMO

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Canais de Cálcio Tipo L/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Ancoragem à Quinase A , Motivos de Aminoácidos , Sequência de Aminoácidos , Transporte Biológico , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/química , Hemaglutininas , Humanos , Dados de Sequência Molecular
17.
Mol Pharmacol ; 61(3): 649-58, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11854446

RESUMO

We have synthesized a novel series of 18 dialkyl 1,4-dihydro-4-(2'alkoxy-6'-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates from anacardic acid, a natural compound found in cashew nut shells, and investigated their blocking action on L- and T-type calcium channels transiently expressed in tSA-201 cells. The IC(50) values for L-type calcium channel block obtained with the series ranged from 1 to approximately 40 microM, with higher affinities being favored by increasing the size of the alkoxy group on the 4-phenyl ring and ester substituent in the 3,5 positions. A detailed analysis of the strongest L-type channel blocker of the series (PPK-12) revealed that block was poorly reversible and mediated an apparent speeding of the time course of inactivation. Moreover, in the presence of PPK-12, the midpoint of the steady state inactivation curve was shifted by 20 mV toward more hyperpolarized potentials, resulting in an increase in blocking efficacy at more depolarized holding potentials. Surprisingly, PPK-12 blocked T- and L-type calcium channels with similar affinities. One of the weakest L-type channel inhibitors (PPK-5) exhibited a T-type channel affinity that was similar to that seen with PPK-12, resulting in a 40-fold selectivity of PPK-5 for T- over L-type channels. Thus, dialkyl 1,4-dihydro-4-(2'alkoxy-6'-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates may serve as excellent candidates for the development of T-type calcium-channel specific blockers.


Assuntos
Bloqueadores dos Canais de Cálcio/síntese química , Canais de Cálcio Tipo T/metabolismo , Nifedipino/síntese química , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Células Cultivadas , Eletrofisiologia , Humanos , Nifedipino/análogos & derivados , Nifedipino/química , Nifedipino/farmacologia , Relação Estrutura-Atividade , Transfecção
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