RESUMO
Neuronal diversity in the cochlea is largely determined by ion channels. Among voltage-gated channels, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open with hyperpolarization and depolarize the cell until the resting membrane potential. The functions for hearing are not well elucidated and knowledge about localization is controversial. We created a detailed map of subcellular location and co-expression of all four HCN subunits across different mammalian species including CBA/J, C57Bl/6N, Ly5.1 mice, guinea pigs, cats, and human subjects. We correlated age-related hearing deterioration in CBA/J and C57Bl/6N with expression levels of HCN1, -2, and -4 in individual auditory neurons from the same cohort. Spatiotemporal expression during murine postnatal development exposed HCN2 and HCN4 involvement in a critical phase of hair cell innervation. The huge diversity of subunit composition, but lack of relevant heteromeric pairing along the perisomatic membrane and axon initial segments, highlighted an active role for auditory neurons. Neuron clusters were found to be the hot spots of HCN1, -2, and -4 immunostaining. HCN channels were also located in afferent and efferent fibers of the sensory epithelium. Age-related changes on HCN subtype expression were not uniform among mice and could not be directly correlated with audiometric data. The oldest mice groups revealed HCN channel up- or downregulation, depending on the mouse strain. The unexpected involvement of HCN channels in outer hair cell function where HCN3 overlaps prestin location emphasized the importance for auditory function. A better understanding may open up new possibilities to tune neuronal responses evoked through electrical stimulation by cochlear implants.
Assuntos
Envelhecimento/metabolismo , Cóclea/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Neurônios/metabolismo , Canais de Potássio/fisiologia , Animais , Gatos , Cóclea/crescimento & desenvolvimento , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Regulação da Expressão Gênica , Cobaias , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neurônios/ultraestrutura , Canais de Potássio/biossíntese , Canais de Potássio/genética , Frações Subcelulares/metabolismoRESUMO
Resistance towards known antimalarial drugs poses a significant problem, urging for novel drugs that target vital proteins in the malaria parasite Plasmodium falciparum. However, recombinant production of malaria proteins is notoriously difficult. To address this, we have investigated two putative K+ channels, PfKch1 and PfKch2, identified in the P. falciparum genome. We show that PfKch1 and PfKch2 and a C-terminally truncated version of PfKch1 (PfKch11-1094) could indeed be functionally expressed in vivo, since a K+-uptake deficient Saccharomyces cerevisiae strain was complemented by the P. falciparum cDNAs. PfKch11-1094-GFP and GFP-PfKch2 fusion proteins were overexpressed in yeast, purified and reconstituted in lipid bilayers to determine their electrophysiological activity. Single channel conductance amounted to 16 ± 1 pS for PfKch11-1094-GFP and 28 ± 2 pS for GFP-PfKch2. We predicted regulator of K+-conductance (RCK) domains in the C-terminals of both channels, and we accordingly measured channel activity in the presence of Ca2+.
Assuntos
Plasmodium falciparum/genética , Canais de Potássio/biossíntese , Proteínas de Protozoários/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Canais de Potássio/genética , Domínios Proteicos , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genéticaRESUMO
The dynamic interaction of DNA methylation and transcription factor binding in regulating spatiotemporal gene expression is essential for embryogenesis, but the underlying mechanisms remain understudied. In this study, using mouse models and integration of in vitro and in vivo genetic and epigenetic analyses, we show that the binding of REST (repressor element 1 (RE1) silencing transcription factor; also known as NRSF) to its cognate RE1 sequences is temporally regulated by non-CpG methylation. This process is dependent on DNA methyltransferase 3B (DNMT3B) and leads to suppression of adult cardiac genes in developing hearts. We demonstrate that DNMT3B preferentially mediates non-CpG methylation of REST-targeted genes in the developing heart. Downregulation of DNMT3B results in decreased non-CpG methylation of RE1 sequences, reduced REST occupancy, and consequently release of the transcription suppression during later cardiac development. Together, these findings reveal a critical gene silencing mechanism in developing mammalian hearts that is regulated by the dynamic interaction of DNMT3B-mediated non-CpG methylation and REST binding.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Coração/embriologia , Miocárdio/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Camundongos , Camundongos Endogâmicos C57BL , Canais de Potássio/biossíntese , Canais de Potássio/genética , Ligação Proteica , Proteínas Repressoras/fisiologia , DNA Metiltransferase 3BRESUMO
Many ion channels, both selective and nonselective, have reentrant pore loops that contribute to the architecture of the permeation pathway. It is a fundamental feature of these diverse channels, regardless of whether they are gated by changes of membrane potential or by neurotransmitters, and is critical to function of the channel. Misfolding of the pore loop leads to loss of trafficking and expression of these channels on the cell surface. Mature tetrameric potassium channels contain an α-helix within the pore loop. We systematically mutated the "pore helix" residues of the channel Kv1.3 and assessed the ability of the monomer to fold into a tertiary reentrant loop. Our results show that pore loop residues form a canonical α-helix in the monomer early in biogenesis and that disruption of tertiary folding is caused by hydrophilic substitutions only along one face of this α-helix. These results provide insight into the determinants of the reentrant pore conformation, which is essential for ion channel function.
Assuntos
Canais de Potássio/biossíntese , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Canais de Potássio/química , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Canais de Potássio/biossíntese , Canais de Potássio/química , Proteínas de Bactérias/genética , Sistema Livre de Células , Cristalografia por Raios X , Técnicas In Vitro , Bicamadas Lipídicas/química , Proteínas de Membrana/genética , Modelos Moleculares , Canais de Potássio/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.
Assuntos
NF-kappa B/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Transcrição Gênica , Animais , Hipóxia Celular/genética , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Humanos , NF-kappa B/genética , Células PC12 , Canais de Potássio/biossíntese , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Sódio/metabolismoRESUMO
KcsA, the bacterial K(+) channel from Streptomyces lividans, is the prototypical model system to study the functional and structural correlations of the pore domain of eukaryotic voltage-gated K(+) channels (Kv channels). It contains all the molecular elements responsible for ion conduction, activation, deactivation and inactivation gating [1]. KcsA's structural simplicity makes it highly amenable for structural studies. Therefore, it is methodological advantageous to produce large amounts of functional and properly folded KcsA in a cost-effective manner. In the present study, we show an optimized protocol for the over-expression and purification of large amounts of high-quality, fully functional and crystallizable KcsA using inexpensive detergents, which significantly lowered the cost of the purification process.
Assuntos
Proteínas de Bactérias , Expressão Gênica , Canais de Potássio , Streptomyces lividans/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Canais de Potássio/biossíntese , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Streptomyces lividans/metabolismoRESUMO
OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.
Assuntos
Canalopatias/genética , Técnicas Eletrofisiológicas Cardíacas , Epilepsia Tipo Ausência/genética , Epilepsia do Lobo Temporal/genética , Frequência Cardíaca/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais de Potássio/genética , Animais , Canalopatias/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas/métodos , Epilepsia Tipo Ausência/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , Masculino , Canais de Potássio/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos WistarRESUMO
BACKGROUND: A number of studies have shown that ethanol (EtOH) activates dopamine neurocircuitries and is self-administered into the ventral tegmental area (VTA) of the rat brain. In vitro and in silico studies have showed that hyperpolarization-activated cyclic nucleotide-gated (HCN) ionic channels on VTA dopamine neurons may constitute a molecular target of EtOH; however, there is no in vivo evidence supporting this assumption. METHODS: Wistar-derived University of Chile Drinking (UChB) rats were microinjected into the VTA with a lentiviral vector coding for rat HCN-2 ionic channel or a control vector. Four days after vector administration, daily voluntary EtOH intake was assessed for 30 days under a free-access paradigm to 5% EtOH and water. After EtOH consumption studies, the effect of HCN-2 overexpression was also assessed on EtOH-induced conditioned place preference (CPP); EtOH-induced locomotion, and EtOH-induced dopamine release in the nucleus accumbens (NAcc). RESULTS: Rats microinjected with the HCN-2 coding vector into the VTA showed (i) a ~2-fold increase in their voluntary EtOH intake compared to control animals, (ii) lentiviral-HCN-2-treated animals also showed an increased CPP to EtOH (~3-fold), (iii) a significant higher locomotor activity (~2-fold), and (iv) increased dopamine release in NAcc upon systemic administration of EtOH (~2-fold). CONCLUSIONS: Overexpression of HCN-2 ionic channel in the VTA of rats results in an increase in voluntary EtOH intake, EtOH-induced CPP, locomotor activity, and dopamine release in NAcc, suggesting that HCN levels in the VTA are relevant for the rewarding properties of EtOH.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Etanol/administração & dosagem , Regulação da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , Canais de Potássio/biossíntese , Recompensa , Área Tegmentar Ventral/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Ratos , Ratos Wistar , Autoadministração , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
One of the main strategies for cancer therapy is to use tyrosine kinase inhibitors for inhibiting tumor proliferation. Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) of these drugs. We report here that a widely used selective inhibitor of Src tyrosine kinases, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), can inhibit and prevent ß-adrenergic stimulation of cardiac pacemaker activity. First, in dissected rat sinus node, PP2 inhibited and prevented the isoproterenol-induced increase of spontaneous beating rate. Second, in isolated rat sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (traditionally called cardiac pacemaker current, I(f)) by negatively shifting the activation curve and decelerating activation kinetics. Third, in isolated rat sinus node myocytes, PP2 decreased the Src kinase activity, the cell surface expression, and tyrosine phosphorylation of hyperpolarization-activated, cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. Finally, in human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed the enhancement of HCN4 channels by isoproterenol and inhibited 573x, a cyclic adenosine momophosphate-insensitive human HCN4 mutant. These results demonstrated that inhibition of Src kinase activity in heart by PP2 decreased and prevented ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the main channel protein that controls the heart rate.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Pirimidinas/farmacologia , Nó Sinoatrial/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Animais , Células Cultivadas , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , Proteínas Musculares/biossíntese , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Canais de Potássio/biossíntese , Ratos , Ratos Sprague-Dawley , Nó Sinoatrial/enzimologia , Quinases da Família src/metabolismoRESUMO
The dorsal and ventral regions of the hippocampus perform different functions. Whether the integrative properties of hippocampal cells reflect this heterogeneity is unknown. We focused on dendrites where most synaptic input integration takes place. We report enhanced backpropagation and theta resonance and decreased summation of synaptic inputs in ventral versus dorsal CA1 pyramidal cell distal dendrites. Transcriptional Kv4.2 down-regulation and post-transcriptional hyperpolarization-activated cyclic AMP-gated channel (HCN1/2) up-regulation may underlie these differences, respectively. Our results reveal differential dendritic integrative properties along the dorso-ventral axis, reflecting diverse computational needs.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/biossíntese , Dendritos/metabolismo , Regulação para Baixo/fisiologia , Canais Iônicos/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Canais de Potássio/biossíntese , Células Piramidais/metabolismo , Canais de Potássio Shal/biossíntese , Regulação para Cima/fisiologia , Animais , Dendritos/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Especificidade de Órgãos , Células Piramidais/citologia , Ratos , Transcrição Gênica/fisiologiaRESUMO
Genomic imprinting is an epigenetic chromosomal modification in the gamete or zygote causing preferential expression of a specific parental allele in somatic cells of the offspring. We and others have identified three imprinted human genes on 11p15.5, IGF2, H19, and p57KIP2, although the latter gene is separated by 700 kb from the other two, and it is unclear whether there are other imprinted genes within this large interval. We previously mapped an embryonal tumour suppressor gene to this region, as well as five balanced germline chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS), a condition characterized by prenatal overgrowth and cancer. We isolated the upstream exons of the previously identified gene KVLQT1, which causes the familial cardiac defect long-QT (LQT) syndrome. We found that KVLQT1 spans much of the interval between p57KIP2 and IGF2, and that it is also imprinted. We demonstrated that the gene is disrupted by chromosomal rearrangements in BWS patients, as well as by a balanced chromosomal translocation in an embryonal rhabdoid tumour. Furthermore, the lack of parent-of-origin effect in LQT syndrome appears to be due to relative lack of imprinting in the affected tissue, cardiac muscle, representing a novel mechanism for variable penetrance of a human disease gene.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 11/genética , Deleção de Genes , Impressão Genômica , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 11/ultraestrutura , Epistasia Genética , Feminino , Proteínas Fetais/biossíntese , Proteínas Fetais/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Humanos , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Neoplasias Renais/genética , Masculino , Dados de Sequência Molecular , Síndromes Neoplásicas Hereditárias/genética , Especificidade de Órgãos , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Canais de Potássio/biossíntese , Translocação Genética/genética , Tumor de Wilms/genéticaRESUMO
In contrast with pathological hypertrophy, exercise-induced physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk. Recent studies have shown that increased cardiac-specific expression of phosphoinositide-3-kinase-α (PI3Kα), the key mediator of physiological hypertrophy, results in transcriptional upregulation of ion channel subunits in parallel with the increase in myocyte size (cellular hypertrophy) and the maintenance of myocardial excitability. The experiments here were undertaken to test the hypothesis that Akt1, which underlies PI3Kα-induced cellular hypertrophy, mediates the effects of augmented PI3Kα signaling on the transcriptional regulation of cardiac ion channels. In contrast to wild-type animals, chronic exercise (swim) training of mice (Akt1(-/-)) lacking Akt1 did not result in ventricular myocyte hypertrophy. Ventricular K(+) current amplitudes and the expression of K(+) channel subunits, however, were increased markedly in Akt1(-/-) animals with exercise training. Expression of the transcripts encoding inward (Na(+) and Ca(2+)) channel subunits were also increased in Akt1(-/-) ventricles following swim training. Additional experiments in a transgenic mouse model of inducible cardiac-specific expression of constitutively active PI3Kα (icaPI3Kα) revealed that short-term activation of PI3Kα signaling in the myocardium also led to the transcriptional upregulation of ion channel subunits. Inhibition of cardiac Akt activation with triciribine in this (inducible caPI3Kα expression) model did not prevent the upregulation of myocardial ion channel subunits. These combined observations demonstrate that chronic exercise training and enhanced PI3Kα expression/activity result in transcriptional upregulation of myocardial ion channel subunits independent of cellular hypertrophy and Akt signaling.
Assuntos
Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Fosfatidilinositol 3-Quinase/metabolismo , Condicionamento Físico Animal , Canais de Potássio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Cardiomegalia , Coração/fisiologia , Ventrículos do Coração/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/biossíntese , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Ribonucleosídeos/farmacologia , Regulação para CimaRESUMO
Large conductance, Ca(2+)-activated K channel proteins are involved in a wide range of physiological activities, so there is considerable interest in the pharmacology of large conductance calcium-activated K (BK) channels. One potent activator of BK channels is mallotoxin (MTX), which produces a very large hyperpolarizing shift of the voltage gating of heterologously expressed BK channels and causes a dramatic increase in the activity of BK channels in human smooth muscle cells. However, we found that MTX shifted the steady-state activation of BK channels in native parotid acinar cells by only 6 mV. This was not because the parotid BK isoform (parSlo) is inherently insensitive to MTX as MTX shifted the activation of heterologously expressed parSlo channels by 70 mV. Even though MTX had a minimal effect on steady-state activation of parotid BK channels, it produced an approximate 2-fold speeding of the channel-gating kinetics. The BK channels in parotid acinar cells have a much more hyperpolarized voltage activation range than BK channels in most other cell types. We found that this is probably attributable to an accessory protein, LRRC26, which is expressed in parotid glands: expressed parSlo + LRRC26 channels were resistant to the actions of MTX. Another class of BK activators is the benzimidazalones that includes 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). Although the LRRC26 accessory protein strongly inhibited the ability of MTX to activate BK channels, we found that it had only a small effect on the action of NS-1619 on BK channels. Thus, the LRRC26 BK channel accessory protein selectively alters the pharmacology of BK channels.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Proteínas de Neoplasias/fisiologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Glândula Parótida/efeitos dos fármacos , Glândula Parótida/metabolismo , Técnicas de Patch-Clamp , Picrotoxina/análogos & derivados , Picrotoxina/toxicidade , Canais de Potássio/agonistas , Canais de Potássio/biossínteseRESUMO
The hyperpolarization-activated cation current I(h) is an important regulator of neuronal excitability and may contribute to the properties of the dentate gyrus granule (DGG) cells, which constitute the input site of the canonical hippocampal circuit. Here, we investigated changes in I(h) in DGG cells in human temporal lobe epilepsy (TLE) and the rat pilocarpine model of TLE using the patch-clamp technique. Messenger-RNA (mRNA) expression of I(h)-conducting HCN1, 2 and 4 isoforms was determined using semi-quantitative in-situ hybridization. I(h) density was â¼1.8-fold greater in DGG cells of TLE patients with Ammon's horn sclerosis (AHS) as compared to patients without AHS. The magnitude of somatodendritic I(h) was enhanced also in DGG cells in epileptic rats, most robustly during the latent phase after status epilepticus and prior to the occurrence of spontaneous epileptic seizures. During the chronic phase, I(h) was increased â¼1.7-fold. This increase of I(h) was paralleled by an increase in HCN1 and HCN4 mRNA expression, whereas HCN2 expression was unchanged. Our data demonstrate an epilepsy-associated upregulation of I(h) likely due to increased HCN1 and HCN4 expression, which indicate plasticity of I(h) during epileptogenesis and which may contribute to a compensatory decrease in neuronal excitability of DGG cells.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/biossíntese , Giro Denteado/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Canais de Potássio/biossíntese , Animais , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/síntese química , Giro Denteado/metabolismo , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/terapia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Pilocarpina/farmacologia , Canais de Potássio/síntese química , Ratos , Regulação para CimaRESUMO
OBJECTIVE: To assess the functional role of Hyperpolarization-activated cyclic nucleotide-gated gated channel (HCN) subtypes in the aging bladder phenotype characterized by diminished bladder volume sensation (BVS) with or without the detrusor instability (DI). METHODS: Expression of HCN subtypes was examined by quantitative RT-PCR and Western blot in aged male Fisher 344 rats (n = 15) and young rats (n = 15). Nocturnal urination and awake cystometry (CMG) were assessed in presence and absence of a steady state HCN channel blockade achieved with daily oral gavage of vehicle or Ivabradine (HCN blocker) 6 mg/kg for 7 days. RESULTS: The association of BVS with the age-related downregulation (~30%) of cAMP sensitive HCN1, HCN2 subtypes, and (~50%) upregulation of cAMP insensitive HCN3 subtype is evinced by the doubling in the mean urine volume of nocturnal voids (0.82 ± 0.22 mL vs 0.41 ± 0.12 mL; n = 10; p < 0.05) predicting an age-related rise in the micturition volume threshold (p < 0.0001) in CMG, which is raised further by Ivabradine treatment (p < 0.0005). Ivabradine also doubled non-voiding contractions (NVC) and maximum voiding pressure (MVP) in young and aged rats, respectively (p < 0.0001) to abolish the age-related, innate two -fold elevation in NVC not accompanied with MVP rise in untreated aged rats (p < 0.005). CONCLUSION: The age-related HCN downregulation is mechanistically linked to the exhibition of aging bladder phenotype with the manifestation of DI following steady state blockade of HCN channels in Ivabradine treated young rats. The amplification of MVP in aged rats mediated by FDA approved Ivabradine hints at potential repurposing opportunity in detrusor underactivity.
Assuntos
Envelhecimento/metabolismo , Regulação da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , Canais de Potássio/biossíntese , Bexiga Inativa/metabolismo , Bexiga Urinária/metabolismo , Envelhecimento/patologia , Animais , Masculino , Ratos , Ratos Endogâmicos F344 , Bexiga Urinária/patologia , Bexiga Inativa/patologiaRESUMO
Hyperpolarization-activated inward currents (I(h)) contribute to neuronal excitability in sensory neurons. Four subtypes of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate I(h), with different activation kinetics and cAMP sensitivities. The aim of the present study was to examine the postnatal development of I(h) and HCN channel subunits in trigeminal ganglion (TG) neurons. I(h) was investigated in acutely dissociated TG neurons from rats aged between postnatal day (P)1 and P35 with whole cell patch-clamp electrophysiology. In voltage-clamp studies, I(h) was activated by a series of hyperpolarizing voltage steps from -40 mV to -120 mV in -10-mV increments. Tail currents from a common voltage step (-100 mV) were used to determine I(h) voltage dependence. I(h) activation was faster in older rats and occurred at more depolarized potentials; the half-maximal activation voltage (V(1/2)) changed from -89.4 mV (P1) to -81.6 mV (P35). In current-clamp studies, blocking I(h) with ZD7288 caused membrane hyperpolarization and increases in action potential half-duration at all postnatal ages examined. ZD7288 also reduced the action potential firing frequency in multiple-firing neurons. Western blot analysis of the TG detected immunoreactive bands corresponding to all HCN subtypes. HCN1 and HCN2 band density increased with postnatal age, whereas the low-intensity HCN3 and moderate-intensity HCN4 bands were not changed. This study suggests that functional I(h) are activated in rat trigeminal sensory neurons from P1 during postnatal development, have an increasing role with age, and modify neuronal excitability.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Canais de Potássio/biossíntese , Células Receptoras Sensoriais/fisiologia , Gânglio Trigeminal/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Western Blotting , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos , Dados de Sequência Molecular , Nociceptividade/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio/fisiologia , Subunidades Proteicas , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismoRESUMO
TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.
Assuntos
Clatrina/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Animais , Western Blotting , Linhagem Celular , Clatrina/genética , Clatrina/isolamento & purificação , Canais de Cátion Regulados por Nucleotídeos Cíclicos/biossíntese , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , DNA/biossíntese , DNA/genética , Eletroforese em Gel de Poliacrilamida , Endocitose , Escherichia coli/metabolismo , Éxons/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Imuno-Histoquímica , Espectrometria de Massas , Proteínas de Membrana/isolamento & purificação , Plasmídeos/genética , Mutação Puntual , Canais de Potássio/biossíntese , Canais de Potássio/genética , Ligação Proteica , Ratos , Frações Subcelulares/metabolismoRESUMO
Mechanisms underlying obesity-related vascular dysfunction are unclear. This study examined the effect of diet-induced obesity on expression and function of large conductance Ca(2+)-activated potassium channel (BK(Ca)) in rat pressurized small resistance vessels with myogenic tone. Male Sprague-Dawley rats fed a cafeteria-style high fat diet (HFD; â¼30% energy from fat) for 16-20 wk were â¼30% heavier than controls fed standard chow (â¼13% fat). Obesity did not alter BK(Ca) α-subunit function or α-subunit protein or mRNA expression in vessels isolated from the cremaster muscle or middle-cerebral circulations. In contrast, BK(Ca) ß(1)-subunit protein expression and function were significantly reduced in cremaster muscle arterioles but increased in middle-cerebral arteries from obese animals. Immunohistochemistry showed α- and ß(1)-subunits were present exclusively in the smooth muscle of both vessels. Cremaster muscle arterioles from obese animals showed significantly increased medial thickness, and media-to-lumen ratio and pressurized arterioles showed increased myogenic tone at 30 mmHg, but not at 50-120 mmHg. Myogenic tone was not affected by obesity in middle-cerebral arteries. The BK(Ca) antagonist iberiotoxin constricted both cremaster muscle and middle-cerebral arterioles from control rats; this effect of iberiotoxin was abolished in cremaster muscle arteries only from obese rats. Diet-induced obesity has contrasting effects on BK(Ca) function in different vascular beds, through differential effects on ß(1)-subunit expression. However, these alterations in BK(Ca) function had little effect on overall myogenic tone, suggesting that the mechanisms controlling myogenic tone can be altered and compensate for altered BK(Ca) expression and function.
Assuntos
Arteríolas/metabolismo , Artérias Cerebrais/metabolismo , Dieta , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Canais de Potássio/biossíntese , Animais , Western Blotting , Gorduras na Dieta/farmacologia , Ingestão de Energia/fisiologia , Frequência Cardíaca/fisiologia , Hiperinsulinismo/etiologia , Hiperinsulinismo/fisiopatologia , Hipertensão/etiologia , Hipertensão/fisiopatologia , Imuno-Histoquímica , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Leptina/sangue , Masculino , Microscopia Eletrônica , Tono Muscular/fisiologia , Músculo Esquelético/irrigação sanguínea , Canais de Potássio/agonistas , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Aumento de Peso/fisiologiaRESUMO
Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 µM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.