RESUMO
The contribution of pharmacologically distinct Ca2+ channels to prepulse-induced facilitation was studied in mouse cerebellar granule cells. Ca2+ channel facilitation was measured as the percentage increase in the whole-cell current recorded during a test pulse before and after it was paired with a positive prepulse. The amount of facilitation was small in recordings made during the first few days in tissue culture but increased substantially after 1 week. L-type channels accounted for the largest proportion of facilitation in 1-week-old cells (60-70%), whereas N-type channels contributed very little (approximately 3%). The toxins omega-agatoxin IVa or omega-conotoxin MVIIC (after block of N-, L-, and P-type channels) each blocked a small percentage of facilitation (approximately 12 and 14%, respectively). Perfusion of cells with GTP-gamma-S enhanced the facilitation of N-type channels, whereas it inhibited of L-type channels. During development in vitro, the contribution of L-type channels to the whole-cell current decreased. Single-channel recordings showed the presence of 10 and 15 pS L-type Ca2+ channels in 1-d-old cells. After 1 week in culture, a approximately 25 pS L-type channel dominated recordings from cell-attached patches. Positive prepulses increased the activity of the 25 pS channel but not of the smaller conductance channels. The expression of Ca(2+) channel facilitation during development may contribute to changes in excitability that allow frequency-dependent Ca(2+) influx during the period of active synaptogenesis
Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Neurônios/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Senescência Celular , Cerebelo/citologia , Di-Hidropiridinas/agonistas , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Camundongos , FosforilaçãoRESUMO
In skeletal muscle, L-type Ca2+ channels act as voltage sensors to control ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum. It has recently been demonstrated that these ryanodine receptors generate a retrograde signal that modifies L-type Ca2+ -channel activity. Here we demonstrate a tight functional coupling between ryanodine receptors and L-type Ca2+ channel in neurons. In cerebellar granule cells, activation of the type-1 metabotropic glutamate receptor (mGluR1) induced a large, oscillating increase of the L-type Ba2+ current. Activation occurred independently of inositol 1,4,5-trisphosphate and classical protein kinases, but was mimicked by caffeine and blocked by ryanodine. The kinetics of this blockade were dependent on the frequency of Ba2+ current stimulation. Both mGluR1 and caffeine-induced increase in L-type Ca2+ -channel activity persisted in inside-out membrane patches. In these excised patches, ryanodine suppressed both the mGluR1- and caffeine-activated L-type Ca2+ channels. These results demonstrate a novel mechanism for Ca2+ -channel modulation in neurons.
Assuntos
Canais de Cálcio/metabolismo , Proteínas Musculares/metabolismo , Neurônios/metabolismo , Animais , Bário/metabolismo , Cafeína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Potenciais da Membrana , Camundongos , Proteínas Musculares/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Proteínas Quinases/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Fosfolipases Tipo C/metabolismoRESUMO
1. We recorded the activity of single L-type Ca2+ channels from cell-attached patches on mouse cerebellar granule cells. The experiments investigated the mechanism of channel reopening at negative membrane potentials following a strong depolarization. 2. L-type channels that reopened following a strong depolarization showed a wide distribution of single-channel conductances, which ranged from 16 to 28 pS in the presence of 90 mM Ba2+. 3. The distribution of the latencies before reopening was fitted as the sum of two exponential components with time constants tau f approximately 1 and tau s approximately 12 ms at -70 mV. Hyperpolarization reduced the time constant of the slower component approximately e-fold per 43 mV, but had no effect on the faster component. 4. Raising the concentration of external Ba2+ reduced the time constant of the slower component of the reopening latency without altering the fast component. The time constant of the slow component was approximately 27 ms in 10 mM Ba2+ and decreased to 12 ms in 90 mM Ba2+ at -70 mV. The relation between the time constant and external Ba2+ saturated with an apparent KD of approximately 20 mM. 5. The distribution of reopening times was best fitted as the sum of two exponential components with time constants tau f approximately 0.5 ms and tau s approximately 4.5 ms at -70 mV. The conditional latencies before reopening into either the short or long open state were indistinguishable. 6. The results are consistent with the idea that a positively charged blocker occludes the pore during depolarization and channels reopen as the blocker dissociates following repolarization to negative potentials.
Assuntos
Canais de Cálcio/fisiologia , Cerebelo/metabolismo , Potenciais da Membrana/fisiologia , Animais , Bário/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
The activity of single L-type Ca2+ channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were concerned with the mechanism by which aminoglycoside antibiotics inhibit ion flow through the channel. Aminoglycosides produced discrete fluctuations in the single-channel current when added to the external solution. The blocking kinetics could be described as a simple bimolecular reaction between an aminoglycoside molecule and the open channel. The blocking rate was found to be increased when either the membrane potential was made more negative or the concentration of external permeant ion was reduced. Both of these effects are consistent with a blocking site that is located within the channel pore. Other features of block, however, were incompatible with a simple pore blocking mechanism. Hyperpolarization enhanced the rate of unblocking, even though an aminoglycoside molecule must dissociate from its binding site in the channel toward the external solution against the membrane field. Raising the external permeant ion concentration also enhanced the rate of unblocking. This latter finding suggests that aminglycoside affinity is modified by repulsive interactions that arise when the pore is simultaneously occupied by a permeant ion and an aminoglycoside molecule.
Assuntos
Antibacterianos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Aminoglicosídeos , Animais , Canais de Cálcio/efeitos dos fármacos , Eletrofisiologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , SoluçõesRESUMO
The activity of single mechanosensitive channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.
Assuntos
Antibacterianos/farmacologia , Canais Iônicos/metabolismo , Mecanorreceptores/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Aminoglicosídeos , Animais , Eletrofisiologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Técnicas de Patch-Clamp , RatosRESUMO
Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either dy/dy or dy/+ myotubes, but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes, but not in wild-type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.
Assuntos
Ativação do Canal Iônico/fisiologia , Músculo Esquelético/citologia , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Animais , Diferenciação Celular/fisiologia , Eletrofisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , XenopusRESUMO
We investigated the mechanisms by which metabotropic glutamate receptors (mGluRs) modulate specific Ca2+ channels in cerebellar granule cells. A large fraction of the current in granule cells is carried by L- and Q-type Ca2+ channels (about 26% each), whereas N- and P-type contribute proportionally less to the global current (9 and 15%, respectively). l-Aminocyclopentane-dicarboxylate (t-ACPD), (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCGI) and (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG], but not L(+)-2-amino-4-phosphonobutyrate (L-AP4) reduced the Ca2+ current amplitude. The t-ACPD-induced inhibition was fully antagonized by (+/-)-methyl-4-carboxyphenylglycine [(+/-)-MCPG] and blocked by pertussis toxin (PTX). These results are consistent with inhibitory response mediated by mGluR2/R3. The use of specific Ca2+ channel blockers provided evidence that mGluR2/R3 inhibited both L- and N-type Ca2+ currents. In PTX-treated cells, Glu or t-ACPD, but not L-CCGI or L-AP4, increased the Ca2+ current. Consistent with the activation of mGluR1, the antagonists (+)-MCPG and (S)-4C3HPG prevented the facilitation of Ca2+ current produced by t-ACPD. The mGluR1-activated facilitation was completely blocked by nimodipine, indicating that L-type Ca2+ currents were selectively potentiated.
Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Técnicas de Cultura , Eletrofisiologia , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Toxina Pertussis , Receptores de Glutamato Metabotrópico/agonistas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We have studied the role of Mg2+ in the inactivation of inwardly rectifying K+ channels in vascular endothelial cells. Inactivation was largely eliminated in Mg(2+)-free external solutions and the extent of inactivation was increased by raising Mg2+o. The dose-response relation for the reduction of channel open probability showed that Mg2+o binds to a site (KD = approximately 25 microM at -160 mV) that senses approximately 38% of the potential drop from the external membrane surface. Analysis of the single-channel kinetics showed that Mg2+ produced a class of long-lived closures that separated bursts of openings. Raising Mg2+o reduced the burst duration, but less than expected for an open-channel blocking mechanism. The effects of Mg2+o are antagonized by K+o in manner which suggests that K+ competes with Mg2+ for the inactivation site. Mg2+o also reduced the amplitude of the single-channel current at millimolar concentrations by a rapid block of the open channel. A mechanism is proposed in which Mg2+ binds to the closed channel during hyperpolarization and prevents it from opening until it is occupied by K+.
Assuntos
Endotélio Vascular/metabolismo , Magnésio/fisiologia , Canais de Potássio/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/fisiologia , Bovinos , Eletrofisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Cinética , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacosRESUMO
1. We examined the activity of single mechanosensitive ion channels in recordings from cell-attached patches on myoblasts, differentiated myotubes and acutely isolated skeletal muscle fibres from wild-type and mdx and dy mutant mice. The experiments were concerned with the role of these channels in the pathophysiology of muscular dystrophy. 2. The predominant form of channel activity recorded with physiological saline in the patch electrode arose from an approximately 25 pS mechanosensitive ion channel. Channel activity was similar in undifferentiated myoblasts isolated from all three strains of mice. By contrast, channel activity in mdx myotubes was approximately 3-4 times greater than in either wild-type or dy myotubes and arose from a novel mode of mechanosensitive gating. 3. Single mechanosensitive channels in acutely isolated flexor digitorum brevis fibres had properties indistinguishable from those of muscle cells grown in tissue culture. The channel open probability in mdx fibres was approximately 2 times greater than the activity recorded from wild-type fibres. The overall level of activity in fibres, however, was roughly an order of magnitude smaller than in myoblasts or myotubes. 4. Histological examination of the flexor digitorum brevis fibres from mdx mice showed no evidence of myonecrosis or regenerating fibres, suggesting that the elevated channel activity in dystrophin-deficient muscle precedes the onset of fibre degeneration. 5. An early step in the dystrophic process of the mdx mouse, which leads to pathophysiological Ca2+ entry, may be an alteration in the mechanisms that regulate mechanosensitive ion channel activity.
Assuntos
Canais Iônicos/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/fisiopatologia , Animais , Membrana Celular/fisiologia , Eletrofisiologia , Laminina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiopatologia , Técnicas de Patch-Clamp , Estimulação Física , PressãoRESUMO
The inhibition of high-threshold Ca2+ channel currents by activated G-proteins was studied in mouse cerebellar granule cells making use of the hydrolysis-resistant GTP analog GTP-gamma-S. When individual granule cells were internally dialyzed with GTP-gamma-S, the high-threshold Ca2+ current decreased to approximately 20% of its initial value within approximately 2 min. The GTP-gamma-S-resistant current was reduced further by the subsequent addition of either omega-conotoxin or dihydropyridine antagonist, indicating that both N- and L-type Ca2+ channels carried the remaining current. Continuous exposure to the dihydropyridine agonist +(S)-202-791 caused a rapid increase in the GTP-gamma-S-resistant current. The L-type current evoked by the agonist subsequently decreased to the level observed prior to adding the drug following a time course similar to the initial inhibition of the total high-threshold current. A second application of the drug at a later time failed to increase the current a second time, indicating a persistent blockade of the agonist-evoked L-current. Pretreating cells with pertussis toxin prevented the initial inhibition of the total whole-cell Ca2+ channel current as well as the subsequent inhibition of the agonist-evoked L-current. The results show that a pertussis toxin-sensitive G-protein produces a persistent inhibition of L-type Ca2+ channels in these central neurons.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Cerebelo/fisiologia , Di-Hidropiridinas/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Neurônios/fisiologia , Peptídeos Cíclicos/farmacologia , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , ômega-Conotoxinas , Animais , Cádmio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica/efeitos dos fármacos , Potenciais Evocados/efeitos dos fármacos , Gadolínio/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cinética , Camundongos , Neurônios/efeitos dos fármacos , Fatores de TempoRESUMO
Recordings of single-channel activity from cerebellar granule cells show that a component of Ca2+ entry flows through L-type Ca2+ channels that are closed at negative membrane potentials following a strong depolarization, but then open after a delay. The delayed openings can be explained if membrane depolarization drives Ca2+ channels into an inactivated state and some channels return to rest through the open state after repolarization. Whole-cell recordings show that the charge carried by Ca2+ during the tail increases as inactivation progresses, whereas the current during the voltage step decreases. Voltage-dependent inactivation may be a general mechanism in central neurons for enhancing Ca2+ entry by delaying it until after repolarization, when the driving force for ion entry is large. Modifying the rate and extent of inactivation would have large effects on Ca2+ entry through those channels that recover from inactivation by passing through the open state.
Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Neurônios/metabolismo , Oxidiazóis , Animais , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cerebelo/citologia , Eletrofisiologia , Potenciais da Membrana , Camundongos , Neurônios/fisiologia , Ácidos Nicotínicos/farmacologia , EstereoisomerismoRESUMO
Single-channel activity was recorded from cell-attached membrane patches on flexor digitorum brevis fibres acutely isolated from normal and mdx mice at different stages of postnatal development. Recordings from cell-attached patches on both normal and mdx fibres were dominated by the activity of mechanosensitive ion channels with a conductance of approximately 17 pS with 110 mM Ba2+ in the patch electrode. In a small fraction of the patches on mdx fibres from young mice, channels showed very high levels of activity. Channel activity recorded from mdx fibres from older mice was higher than in age-matched normal fibres and the level of activity decreased during development. Channel density decreased in normal fibres, whereas it remained relatively constant in mdx fibres, as if channels are down-regulated in normal, but not mdx, fibres during postnatal development. An early step in the dystrophic process may be an alteration of the mechanisms that regulate the expression of functional channels.
Assuntos
Canais de Cálcio/metabolismo , Músculos/metabolismo , Distrofia Muscular Animal/metabolismo , Fatores Etários , Animais , Eletrofisiologia , Camundongos , Camundongos Mutantes , Desenvolvimento Muscular , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Estresse MecânicoRESUMO
1. Granule cells were dissociated from mouse cerebellum and grown in vitro. Currents through single Ca2+ channels were recorded from the cell body with the patch clamp technique. 2. Voltage steps to 0 mV produced brief channel openings with a mean open time of approximately 0.5 ms. The single-channel conductance measured from the amplitude of the single-channel current with 90 mM-Ba2+ in the patch electrode was 22 pS. 3. The probability of Ca2+ channel opening increased with test potentials more positive than -30 mV, with half-activation near 0 mV, and followed the Boltzmann relation for the activation of whole-cell Ca2+ current. 4. Voltage steps to potentials more positive than 0 mV produced more channel activity at the beginning than at the end of the voltage step. The average of the single-channel currents decayed to a non-zero level with a time course similar to that of the whole-cell Ca2+ current. 5. The amplitude as well as the decay of the mean current measured during a test pulse to 0 mV was reduced as the holding potential was made more positive than approximately -90 mV. The change in the open channel probability with holding potential followed the Boltzmann relation which described the inactivation of the whole-cell Ca2+ current. 6. Ca2+ channel activity persisted for over several minutes after excising the patch from the cell body when intracellular cyclic AMP was increased. After patch excision, the number of functional channels decreased to a level where only one channel at a time was active. Ca2+ channel openings appeared as either short bursts at the beginning of the voltage step or long bursts that lasted throughout the pulse. 7. Exposing the cell to the dihydropyridine agonist +(S)-202-791 markedly increased the fraction of sweeps with long openings and produced a non-decaying mean current that was approximately 5 times larger than control. In a fraction of the sweeps, however, long openings occurred more frequently at the beginning than at the end of the voltage step and these produced a decaying mean current. 8. Shifting the holding potential to more positive potentials in the presence of the dihydropyridine agonist preferentially reduced the number of brief openings while sparing the long openings. The amplitude of the mean current was similar to that obtained from the more negative holding potential and there was no change in the fraction of sweeps with long openings that occurred at the beginning of the voltage pulse.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Canais de Cálcio/fisiologia , Cerebelo/fisiologia , Di-Hidropiridinas/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , AMP Cíclico/farmacologia , Estimulação Elétrica , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Currents flowing through single stretch-inactivated ion channels were recorded from cell-attached patches on myotubes from mdx mice. Adding micromolar concentrations of gadolinium to patch electrodes containing normal saline produced rapid transitions in the single-channel current between the fully open and closed states. The kinetics of the current fluctuations followed the predictions of a simple model of open channel block in which the transitions in the current arise from the entry and exit of Gd from the channel pore: histograms of the open and closed times were well fit with single exponentials, the blocking rate depended linearly on the concentration of gadolinium in the patch electrode, and the unblocking rate was independent of the concentration of gadolinium. Hyperpolarizing the patch increased the rate of unblocking (approximately e-fold per 85 mV), suggesting the charged blocking particle can exit the channel into the cell under the influence of the applied membrane field. The rate of blocking was rapid and was independent of the patch potential, consistent with the rate of ion entry into the pore being determined by its rate of diffusion in solution. When channel open probability was reduced by applying suction to the electrode, the blocking kinetics were independent of the extent of inactivation, suggesting that mechanosensitive gating does not modify the structure of the channel pore.
Assuntos
Gadolínio/farmacologia , Canais Iônicos/efeitos dos fármacos , Músculos/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Canais Iônicos/metabolismo , Cinética , Potenciais da Membrana , Camundongos , Camundongos Mutantes , Distrofia Muscular Animal/metabolismo , Estresse MecânicoRESUMO
Recordings of single-channel activity were made from cell-attached patches on cerebellar granule cells from normal and mdx mice. Recordings from mdx granule cells show the activity of ion channels that are open for seconds at negative holding potentials near rest. These channels are permeable to divalent cations and have a conductance of 8-10 pS with either Ca2+ or Ba2+ as the charge carrier in the patch electrode. Under similar recording conditions, channel activity is virtually absent from normal mouse granule cells. The absence of dystrophin in neurons, as well as in skeletal muscle, is associated with an increase in the activity of Ca(2+)- permeable ion channels. Increased channel activity may be an early event leading to pathophysiological accumulation of intracellular Ca2+ in Duchenne muscular dystrophy.
Assuntos
Compostos de Bário , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Cerebelo/fisiopatologia , Cloretos , Neurônios/fisiologia , Animais , Bário/farmacologia , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Cerebelo/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Neurônios/efeitos dos fármacos , Probabilidade , Valores de ReferênciaRESUMO
1. Cells dissociated from mouse cerebellum were grown in vitro. Ca2+ channel currents were recorded from granule cells with the patch-clamp technique under conditions which suppressed currents through Na+ and K+ channels and minimized run-down of current through Ca2+ channels. 2. A strong depolarizing voltage step from a hyperpolarized holding potential produced inward Ca2+ channel current that decayed exponentially to a non-zero level. Inward current decayed to approximately 40% of its peak amplitude (range 20-90%). 3. The inward current increased in amplitude when Ca2+ was replaced with Ba2+ or after raising the concentration of extracellular Ba2+, but the rate of decay of current was unaffected. 4. The current-voltage (I-V) relation showed that peak or sustained current increased with voltage pulses more positive than approximately -30 mV, reached a maximum amplitude near +20 mV and became progressively smaller with larger depolarizations. 5. The tail currents produced after rapidly repolarizing the membrane potential to -70 mV from a positive test pulse decayed along a single exponential time course with a time constant of approximately 0.5 ms. The amplitude of tail current measured at a fixed repolarization potential increased as the pre-pulse was made more positive and reached a maximum with pre-pulses more positive than +40 mV. A plot of normalized amplitude of the tail current as a function of the pre-pulse potential was fitted with a Boltzmann relation with V1/2 = approximately + 8 mV and steepness k = 14 mV. 6. Shifting the holding potential to more positive potentials reduced the amplitude of the Ca2+ channel current elicited by the fixed voltage step and abolished the decay of the inward current. The peak current was normalized to the maximum peak current elicited from a very negative holding potential and plotted as a function of holding potential. The points were fitted with a Boltzmann relation for inactivation with V1/2 = approximately -57 mV and steepness k = 14 mV. 7. The onset of inactivation was studied in two-pulse experiments in which the duration of conditioning pre-pulse was varied. Increasing the duration of a pre-pulse to a fixed potential reduced the peak inward current evoked by the second test pulse. Plotting normalized current as a function of pre-pulse duration showed that inactivation developed along a double exponential time course. Both fast and slow time constants decreased as the pre-pulse potential was made more positive.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cálcio/metabolismo , Cerebelo/metabolismo , Ativação do Canal Iônico/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Células Cultivadas , Di-Hidropiridinas/farmacologia , Eletrofisiologia , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
The blocking actions of Fe2+, Co2+, and Ni2+ on unitary currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCl2 produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarization speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni2+ differs from that produced by Fe2+ and Co2+ because Ni2+ enters the channel approximately 20 times more slowly and exits approximately 50 times more slowly. Ni2+ also differs from the other transition metals because at millimolar concentrations it reduces the amplitude of the unitary current in a concentration-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size.
Assuntos
Canais de Cálcio/metabolismo , Animais , Bário/metabolismo , Sítios de Ligação , Canais de Cálcio/efeitos dos fármacos , Linhagem Celular , Cobalto/metabolismo , Cobalto/farmacologia , Ferro/metabolismo , Ferro/farmacologia , Cinética , Potenciais da Membrana , Níquel/metabolismo , Níquel/farmacologiaRESUMO
We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing alpha, beta, delta, and either gamma or epsilon subunits. gamma- and epsilon-AChRs had identical association rates for binding 125I-alpha-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but epsilon-AChRs had a significantly longer half-time of turnover in the membrane than gamma-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to gamma-, but not epsilon-AChRs. The gamma-AChRs had channel characteristics of embryonic AChRs, whereas the major class of epsilon-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with epsilon-AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of gamma- and epsilon-AChRs.
Assuntos
Músculos/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Sítios de Ligação , Bungarotoxinas/antagonistas & inibidores , Bungarotoxinas/metabolismo , Linhagem Celular , Condutividade Elétrica , Expressão Gênica , Cinética , Camundongos , Músculos/embriologia , Testes de Precipitina , Receptores Colinérgicos/genética , Receptores Colinérgicos/imunologia , Transfecção , Tubocurarina/metabolismo , Tubocurarina/farmacologiaRESUMO
1. Recordings of single-channel activity were made from cell-attached patches on mouse C2 muscle cells at morphologically identifiable stages of myogenesis in vitro. We have identified Ca2(+)-permeable, cation-selective channels that are gated by applying suction to the patch electrode and by changes in membrane potential and have analysed single-channel properties as well as channel expression during myogenesis. 2. Single-channel activity could be detected when the membrane was held at steady negative potentials. With monovalent cations in the electrode, the single-channel current-voltage (i-V) relations were linear. The channel is permeable to Li+, Na+, K+, Rb+ and Cs+, but is not strongly selective among the monovalent cations as judged by measurements of single-channel conductance and reversal potential. 3. With 110 mM of either CaCl2 or BaCl2 as the only inward change carrier, slope conductances were approximately 13 and 24 pS and currents reversed at approximately +22 and +17 mV, respectively. The relative permeability of Ca2+ to K+ calculated from the constant-field equation was PCa/PK = approximately 2. 4. Channel openings occurred as bursts of brief openings and closings separated by much longer closed periods. Closed-time histograms were best fitted with three exponential components, while histograms of burst duration were best fitted with two exponential components, reflecting the short and long bursts in the single-channel records. 5. Applying suction to the patch electrode while recording at steady negative membrane potentials produced channel openings to discrete current levels. Mean channel open probability depended linearly on the square of the applied pressure and was greater at positive membrane potentials. The permeability of the channel to monovalent and divalent cations was indistinguishable from the spontaneous activity recorded at steady negative potentials. 6. Channel activity recorded from cell-attached patches in the absence of applied pressure depended on membrane potential increasing approximately e-fold per 38 mV with depolarization. Analysis of the kinetics of the response to membrane potential showed that the depolarization reduced the duration of the slowest component of the closed-time distribution. 7. The lanthanide cation gadolinium (Gd) reduced the amplitude of the unitary currents in a concentration-dependent manner. The amplitudes of both inward and outward currents were reduced to the same extent suggesting block is voltage-independent. Gd produced half-maximal inhibition of the unitary current at approximately 6 microM.(ABSTRACT TRUNCATED AT 400 WORDS)