RESUMO
Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100-200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.
Assuntos
Cálcio/fisiologia , Citosol/fisiologia , Mitocôndrias/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio , Proteínas de Ligação ao Cálcio/fisiologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Transporte/fisiologia , Compartimento Celular/fisiologia , Células Cromafins , Citosol/metabolismo , Metabolismo Energético , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos com 3 Anéis , Líquido Intracelular/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Técnicas de Patch-Clamp , Ratos , Trocador de Sódio e CálcioRESUMO
Secretion of gonadotropic hormones from pituitary gonadotropes in response to gonadotropin-releasing hormone (GnRH) is essential for regulation of reproductive potential. Gonadotropes from male rats exhibited an unusual form of cellular excitation that resulted from periodic membrane hyperpolarization. GnRH induced an oscillatory release of intracellular Ca2+ via a guanosine triphosphate (GTP) binding protein-coupled phosphoinositide pathway and hyperpolarized the gonadotrope periodically by opening apamin-sensitive Ca(2+)-activated K+ (SK) channels. Each hyperpolarization was terminated by firing of a few action potentials that may result from removal of inactivation from voltage-gated Na+ and Ca2+ channels.
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/fisiologia , Adeno-Hipófise/fisiologia , Animais , Apamina/farmacologia , Células Cultivadas , Proteínas de Ligação ao GTP/fisiologia , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/fisiologia , Potenciais da Membrana , Periodicidade , Adeno-Hipófise/citologia , Canais de Potássio/fisiologia , RatosRESUMO
In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) induces the rhythmic release of Ca2+ from an inositol 1,4,5-trisphosphate (IP3)-sensitive store. Simultaneous measurement of the concentration of cytosolic free Ca2+ ([Ca2+]i) and exocytosis in single identified gonadotropes showed that each elevation of [Ca2+]i induced a burst of exocytosis. These phenomena were largely suppressed by buffering of [Ca2+]i but persisted in the absence of extracellular Ca2+. Activation of voltage-gated Ca2+ channels by brief depolarizations seldom supplied enough Ca2+ for exocytosis, but [Ca2+]i elevations induced by photolysis of caged IP3 did trigger exocytosis, confirming that GnRH-stimulated gonadotropic hormone secretion is closely coupled to intracellular Ca2+ release. Agonist-induced oscillations of [Ca2+]i in secretory cells may be a mechanism to optimize the secretory output while avoiding the toxic effects of sustained elevation of [Ca2+]i.
Assuntos
Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Hipófise/fisiologia , Potenciais de Ação , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/fisiologia , Eletrofisiologia , Hormônio Liberador de Gonadotropina/administração & dosagem , Inositol 1,4,5-Trifosfato/farmacologia , Masculino , Periodicidade , Fotólise , Hipófise/efeitos dos fármacos , Hipófise/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Local recycling of synaptic vesicle membrane at nerve terminals is necessary to maintain a readily releasable pool of transmitter. To what extent are the dynamics of vesicle recycling subject to modulation? We examined the influence of presynaptic GABA(B) receptors on vesicle dynamics at single synapses using optical imaging of FM1-43 in cultured rat hippocampal neurons. The kinetics of FM1-43 destaining indicate that synapses from a single neuron have a unimodal distribution of release probabilities, and GABA(B)-mediated inhibition occurs uniformly at all sites. Electrical and optical recordings from single cells show that the inhibition of excitatory transmission is entirely accounted for by a rapidly reversible reduction of exocytosis. In contrast, GABA(B) receptors do not alter the rate or extent of endocytosis.
Assuntos
Baclofeno/farmacologia , Hipocampo/fisiologia , Neurônios/fisiologia , Receptores de GABA-B/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Células Cultivadas , Endocitose , Potenciais Evocados/efeitos dos fármacos , Exocitose , Corantes Fluorescentes , Cinética , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Compostos de Piridínio , Compostos de Amônio Quaternário , Ratos , Espectrometria de Fluorescência , Sinapses/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacosRESUMO
We studied inhibition of N-type Ca2+ channels in rat superior cervical ganglion neurons by substance P (SP) and somatostatin-14 (Som). In whole-cell clamp, 70 of 82 acutely dissociated neurons showed inhibition (mean 37%) by 500 nM SP, and 54 of 61 showed inhibition by 240 nM Som (mean 57%). Pertussis toxin (PTX) blocked Som but not SP inhibition; intracellular dialysis with 2 mM GDP-beta-S attenuated inhibition with either peptide. Inhibition was voltage dependent with Som but not with SP. Neurokinin A (1 microM) or B was without effect, implicating NK1 tachykinin receptors. In cell-attached patches with bath-applied drugs, to test for a diffusible messenger, inhibition by SP or Som was only 8%. Thus, SP signaling is voltage independent and PTX insensitive; Som inhibition is voltage dependent and PTX sensitive; and both are membrane delimited.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Gânglios Simpáticos/citologia , Neurônios/fisiologia , Somatostatina/farmacologia , Substância P/farmacologia , Animais , Canais de Cálcio/fisiologia , Diálise , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Condutividade Elétrica , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Toxina Pertussis , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-2 , Receptores de Neurotransmissores/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Tionucleotídeos/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Agonist-induced suppression of current in voltage-gated Ca2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca2+ current-one fast and membrane delimited, the other slow and acting via a diffusible second messenger. We now show that the fast pathway is sensitive mainly to pertussis toxin and shifts the gating of Ca2+ channels to more positive voltages (voltage dependent). The slow pathway is pertussis toxin insensitive and depresses currents at all test potentials (voltage independent). Muscarinic agonists may also activate a pertussis toxin-insensitive fast pathway. alpha-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.
Assuntos
Canais de Cálcio/fisiologia , Neurônios/fisiologia , Toxina Pertussis , Sistema Nervoso Simpático/fisiologia , Fatores de Virulência de Bordetella/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Eletrofisiologia , Proteínas de Ligação ao GTP/fisiologia , Ativação do Canal Iônico/fisiologia , Masculino , Norepinefrina/farmacologia , Oxotremorina/farmacologia , Ratos , Ratos Endogâmicos , Receptores Muscarínicos/fisiologiaRESUMO
Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.
Assuntos
Canais de Cálcio/fisiologia , Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Receptores Muscarínicos/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Condutividade Elétrica , Masculino , Norepinefrina/farmacologia , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Adrenérgicos alfa/fisiologia , Receptores Muscarínicos/efeitos dos fármacosRESUMO
Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.
Assuntos
Canais de Cálcio/fisiologia , Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Receptores Adrenérgicos alfa/fisiologia , Receptores Muscarínicos/fisiologia , Sistemas do Segundo Mensageiro , Animais , Ácido Egtázico/farmacologia , Condutividade Elétrica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Norepinefrina/farmacologia , Oxotremorina/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacosRESUMO
Cytosolic Ca2+ (Ca2+c) clearance from adrenal chromaffin cells was studied by whole-cell patch clamp and indo-1 Ca2+ photometry after influx of Ca2+ through voltage-dependent Ca2+ channels. We isolated the rates of Ca2+c clearance by several mechanisms using combinations of the following agents (with their expected targets): Li+ or TEA substituted for Na+ (Na(+)-Ca2+ exchange), 1 mM La3+ applied after the depolarization (Na(+)-Ca2+ exchange and plasma membrane Ca(2+)-ATPase), 1 microM thapsigargin (pumping into reticular stores), and 2 microM carbonyl cyanide m-chlorophenylhydrazone (uptake into mitochondria). Remarkably, whenever [Ca2+]c rose above approximately 500 nM, Ca2+c clearance by mitochondria exceeded clearance by either Na(+)-Ca2+ exchange or the Ca2+ pumps of the plasma and reticular membranes. As [Ca2+]c fell again, Ca2+ reemerged from mitochondria, prolonging the final return to basal levels.
Assuntos
Medula Suprarrenal/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Mitocôndrias/fisiologia , Medula Suprarrenal/citologia , Medula Suprarrenal/embriologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico Ativo/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Linhagem da Célula , Células Cultivadas , Líquido Intracelular/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lantânio/farmacologia , Lítio/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Crista Neural , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio , Terpenos/farmacologia , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologia , TapsigarginaRESUMO
We characterized inhibition of N-type Ca2+ and M current K+ channels in rat superior cervical ganglion neurons by angiotensin II (angioII) using the patch clamp. Of 120 neurons, 97 showed inhibition of ICa (mean 32%), which was slow in onset and very slow to reverse under whole-cell recording conditions. This inhibition was blocked by the AT1 receptor antagonist losartan, attenuated by inclusion of 2 mM GDP-beta-S in the pipette, mostly pertussis toxin insensitive, half-sensitive to N-ethylmaleimide, and wholly voltage independent. With 20 mM instead of 0.1 mM BAPTA in the pipette, the inhibition was strongly attenuated; however, we detected no angioII-induced [Ca2+]i signal using the fluorescent indicator indo-1. IBa from cell-attached patches was reduced by bath-applied angioII (mean 33%), suggesting use of a diffusible cytoplasmic messenger. M currents were inhibited by angioII in 8 of 11 neurons (mean 50%) cultured overnight. Hence, a second agonist, angioII, may share the slow, second messenger-utilizing, pertussis toxin-insensitive signaling pathway used by muscarinic agonists.
Assuntos
Angiotensina II/farmacologia , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Canais Iônicos/fisiologia , Neurônios/fisiologia , Gânglio Cervical Superior/fisiologia , Antagonistas de Receptores de Angiotensina , Animais , Compostos de Bifenilo/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Etilmaleimida/farmacologia , Corantes Fluorescentes , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Indóis , Canais Iônicos/efeitos dos fármacos , Cinética , Losartan , Potenciais da Membrana/efeitos dos fármacos , Microscopia de Fluorescência , Neurônios/efeitos dos fármacos , Toxina Pertussis , Ratos , Ratos Sprague-Dawley , Sistemas do Segundo Mensageiro , Transdução de Sinais/efeitos dos fármacos , Somatostatina/farmacologia , Tetrazóis/farmacologia , Tionucleotídeos/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Exocytosis and the cell-averaged cytosolic [Ca2+], [Ca2+]i, were tracked in single gonadotrophs. Cells released 100 granules/s at 1 microM = [Ca2+]i when gonadotropin-releasing hormone (GnRH) activated IP3-mediated Ca2+ release from internal stores, but only 1 granule/s when [Ca2+]i was raised uniformly to 1 microM by other means. Strong exocytosis was then seen only at higher [Ca2+]i (half-maximal at 16 microM). Parallel second messengers did not contribute to GnRH-induced exocytosis, because IP3 alone was as effective as GnRH, and because even GnRH failed to trigger rapid exocytosis when the [Ca2+]i rise was blunted by EGTA. When [Ca2+]i was released from stores, exocytosis depended on [Ca2+]i rising rapidly, as if governed by Ca2+ flux into the cytosol. We suggest that IP3 releases Ca2+ selectively from subsurface cisternae, raising [Ca2+] near exocytic sites 5-fold above the cell average.
Assuntos
Cálcio/metabolismo , Grânulos Citoplasmáticos/metabolismo , Exocitose , Hormônio Liberador de Gonadotropina/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Adeno-Hipófise/fisiologia , Acetatos/farmacologia , Animais , Quelantes/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Citosol/metabolismo , Ácido Egtázico/farmacologia , Etilenodiaminas/farmacologia , Técnicas In Vitro , Cinética , Potenciais da Membrana , Técnicas de Patch-Clamp , Fotólise , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/ultraestrutura , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The hypothesis that acetylcholine, substance P, and LHRH suppress M-current by activating phospholipase C was tested. Each agonist caused turnover of phosphoinositide, as measured by release of inositol phosphates, and a modest transient rise in intracellular free Ca2+ ([ Ca2+]i), as determined with fura-2. Active phorbol esters depressed M-current only 50% and did not prevent further suppression by LHRH. M-current, its control by agonists, and its depression by phorbol esters were not affected by adding inositol trisphosphate or Ca2+ buffers with high or low Ca2+ to the whole-cell, voltage-clamp pipette. We conclude that phospholipase C activation does occur but does not mediate the suppression of M-current by agonists. Caffeine produced large [Ca2+]i transients and acted as an agonist to suppress M-current.
Assuntos
Acetilcolina/farmacologia , Cálcio/metabolismo , Gânglios Simpáticos/fisiologia , Hormônio Liberador de Gonadotropina/farmacologia , Fosfatos de Inositol/metabolismo , Neurônios/fisiologia , Fosfatidilinositóis/metabolismo , Substância P/farmacologia , Animais , Atropina/farmacologia , Benzofuranos , Condutividade Elétrica , Ativação Enzimática , Corantes Fluorescentes , Fura-2 , Gânglios Simpáticos/efeitos dos fármacos , Técnicas In Vitro , Muscarina/farmacologia , Neurônios/efeitos dos fármacos , Ranidae , Fosfolipases Tipo C/metabolismoRESUMO
Neurotransmitters acting through G-protein-coupled receptors change the electrical excitability of neurons. Activation of receptors can affect the voltage dependence, the speed of gating, and the probability of opening of various ion channels, thus changing the computational state and outputs of a neuron. Each cell expresses many kinds of receptors, and uses several intracellular signaling pathways to modulate channel function in different ways. It has become possible to dissect these pathways by combining pharmacological and biophysical experiments. Recent patch-clamp work in sympathetic neurons will be summarized to illustrate the mechanisms underlying modulation and its significance.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/agonistas , Proteínas de Ligação ao GTP/fisiologia , Gânglios Simpáticos/fisiologia , Fatores de Virulência de Bordetella/farmacologia , Animais , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Ratos , Sistemas do Segundo MensageiroRESUMO
Mitochondria, the metabolic powerhouses of the cell, can sequester and release large amounts of Ca2+. This import and export of Ca2+ helps to adjust energy production to cellular needs. Recent advances show that mitochondrial Ca2+ fluxes play a major role in normal Ca2+ signaling.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/fisiologia , Mitocôndrias/fisiologia , Transdução de Sinais/fisiologia , Potenciais da Membrana/fisiologia , Mitocôndrias/químicaRESUMO
The rat cell line FR3T3 was transformed with the retroviral oncogenes v-myc or v-src, with the DNA tumor viruses SV40 or bovine papilloma virus strain 1 (BPV-1) or with the 69% transforming region of BPV-1. The transformants were compared with the uncloned parental line for their susceptibility to the lytic effect and to the replication of MVMp, an autonomous parvovirus. Expression of v-myc and v-src proteins and of SV40 large T antigen correlated with a greater cell susceptibility to MVMp-induced killing. Thus, the expression of both cytoplasmic and nuclear oncogene products may sensitize rat fibroblasts to MVMp. In contrast, cell lines transformed by BPV-1, including highly tumorigenic and tumor-derived clones, were on the average as resistant as the parental cell line to MVMp infection. A similar resistance to MVMp-induced killing was displayed by BPV-1-transformed NIH3T3 cells. However, supertransformation of one of the BPV-1-transformants by the human EJ-Harvey ras-1 oncogene, known to sensitize FR3T3 and NIH3T3 cells, correlated with an increase in susceptibility to MVMp. Therefore, the failure of BPV-1 transformation to sensitize murine cells to parvoviral attack may be ascribed to the tumor virus rather than to the cells undergoing transformation. Hence, cell sensitization to MVMp appears to be oncogene-specific and cannot be taken as an absolute correlative with neoplastic transformation.
Assuntos
Transformação Celular Neoplásica , Oncogenes , Parvoviridae/patogenicidade , Animais , Papillomavirus Bovino 1/genética , Replicação do DNA , Amplificação de Genes , Parvoviridae/genética , Fenótipo , Ratos , Vírus 40 dos Símios/genética , Replicação ViralRESUMO
Carbon-fiber amperometry detects oxidizable molecules released by exocytosis. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gonadotropes, cultured cerebellar granule cells, and yeast with high concentrations of dopamine (DA) and observed spontaneous and evoked quantal release of DA by amperometry. The rate of detectable spontaneous amperometric events was used as a measure of loading in AtT-20 cells. With 70 mm DA in the bath, loading was complete within 40 min. Cytoplasmic accumulation preceded vesicular loading. Loading decreased proportionally as the bath DA concentration was lowered. Loading rates were similar at 37 and 25 degrees C and much slower at 15 degrees C. Loading was blocked by bafilomycin A(1), a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane DA transporter. Other cells were tested. Spontaneous quantal events became more frequent and evoked events became larger and more frequent when PC12 cells were loaded with DA. Fluid-phase loading of neurons by short stimulation in DA solutions seemed selective for the synaptic vesicles. Thus, many cell types can be loaded with DA to study spontaneous and evoked exocytosis. The amine molecules enter these cells passively and may become concentrated in acidic vesicles by protonation.
Assuntos
Eletroquímica/métodos , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Neurotransmissores/metabolismo , Vesículas Secretórias/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Citoplasma/metabolismo , Dopamina/metabolismo , Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina , Relação Dose-Resposta a Droga , Eletroquímica/instrumentação , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Camundongos , Neurotransmissores/farmacologia , Oxirredução/efeitos dos fármacos , Células PC12 , Hipófise/citologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Inibidores da Bomba de Prótons , Ratos , Ratos Sprague-Dawley , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMO
Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K(+) current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M(1) muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca(2+) transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca(2+) signals were abolished by the combined use of strong intracellular Ca(2+) buffers, an inhibitor of IP(3) receptors, and thapsigargin to deplete Ca(2+) stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca(2+) as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.
Assuntos
Neurônios/química , Neurônios/fisiologia , Canais de Potássio/genética , Receptores Muscarínicos/genética , Animais , Antracenos/farmacologia , Atropina/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Quelantes/farmacologia , Clonagem Molecular , Citoplasma/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/fisiologia , Humanos , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Masculino , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Neurônios/citologia , Oxotremorina/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Estaurosporina/farmacologia , Gânglio Cervical Superior/citologia , Tetraetilamônio/farmacologia , Tapsigargina/farmacologiaRESUMO
Ionic fluxes in Na channels of myelinated axons show ionic competition, block, and deviations from simple flux independence. These phenomena are particularly evident when external Na+ ions are replaced by other permeant or impermeant ions. The observed currents require new flux equations not based on the concepts of free diffusion. A specific permeability model for the Na channel is developed from Eyring rate theory applied to a chain of saturable binding sites. There are four energy barriers in the pore and only one ion is allowed inside at a time. Deviations from independence arise from saturation. The model shows that ionic permeability ratios measured from zero-current potentials can differ from those measured from relative current amplitudes or conductances. The model can be fitted to experiments with various external sodium substitutes by varying only two parameters: For each ion the height of the major energy barrier (the selectivity filter) determines the biionic zero-current potential and the depth of the energy well (binding site) just external to that barrier then determines the current amplitudes. Voltage clamp measurements with myelinated nerve fibers are given showing numerous examples of deviations from independence in ionic fluxes. Strong blocks of ionic currents by guanidinium compounds and Tl+ ions are fitted by binding within the channel with apparent dissociation constants in the range 50-122 mM. A small block with high Na+ concentrations can be fitted by Na+ ion binding with a dissociation constant of 368 mM. The barrier model is given a molecular interpretation that includes stepwise dehydration of the permeating ion as it interacts with an ionized carboxylic acid.
Assuntos
Modelos Biológicos , Fibras Nervosas Mielinizadas/metabolismo , Sódio/metabolismo , Animais , Anuros , Sítios de Ligação , Permeabilidade da Membrana Celular , Cloro/metabolismo , Eletrofisiologia , Potenciais da Membrana , Potássio/metabolismoRESUMO
The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na(+) approximately Li(+) > Tl(+) > K(+). The ratio P(K)/P(Na) is 1/12. The permeabilities to Rb(+), Cs(+), Ca(++), and Mg(++) are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E(Na) differs significantly from the Nernst potential for Na(+) in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.