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H D Lux

Publications and source records attributed to H D Lux.

At least 55 records · Page 3Linked to original sources
PubMed1989

Glial cells of the oligodendrocyte lineage express proton-activated Na+ channels.

Neurons and oligodendrocytes, but not type I astrocytes and Schwann cells, generate large Na+ currents in response to a step increase of [H+]. Proton-activated Na+ channels are the first cationic channels expressed in neuronal precursor cells from the mammalian brain. Glial precursor cells cultured from mouse brain are also capable of generating Na+ currents in response to step acidification (INa(H]. With further development along the oligodendrocyte lineage, this property is retained, whereas voltage-activated Na+ and K+ currents disappear. Comparing INa(H) of oligodendrocytes with INa(H) of their precursor cells did not reveal a difference in current amplitude, suggesting a higher density of INa(H) channels on the (smaller) precursor cells. The properties of INa(H) in glial precursor cells and oligodendrocytes are similar to those of neurons, with respect to activation conditions, time course, and the effect of extracellular Ca2+ concentrations. The results are consistent with previous observations which showed that oligodendrocytes partially preserve their chemically activated, but completely lose their voltage-activated, ion channels.

Animals↗
PubMed1989

Opposing effects of acetylcholine on the two classes of voltage-dependent calcium channels in hippocampal neurons.

Acetylcholine (Ach) was tested for its effect on calcium currents in primary cultures of embryonic rat hippocampal neurons. Ach reversibly depressed, in a dose-dependent way, the high voltage activated (HVA) Ca currents. The effect was antagonized by atropine. Our results suggest that a pertussis toxin (PTX)-sensitive GTP binding protein (G-protein) is involved in the signal transduction mechanism between the Ach receptor and the HVA Ca channel. Activating rather than depressive effects of Ach were observed on the low voltage-activated component of Ca currents. This effect was also antagonized by atropine but is not mediated by a PTX-sensitive G-protein.

Acetylcholine↗
PubMed1989

Direct modulation of voltage-dependent calcium channels by muscarinic activation of a pertussis toxin-sensitive G-protein in hippocampal neurons.

Acetylcholine (Ach) reversibly reduces the high voltage-activated (HVA) calcium (Ca) current in hippocampal neurons. Pretreatment of the cells with pertussis toxin (PTX) abolishes the Ach effect, suggesting that PTX-sensitive GTP-binding regulatory proteins (G-proteins) are involved in the signal transduction mechanism that links Ach receptor activation to inhibition of Ca channel activity. This effect is mimicked by intracellular application of the nonhydrolyzable GTP analog GTP gamma S. Intracellular application of purified G-proteins restored the response to Ach in PTX-treated cells. Furthermore, Ach inhibits the Ca current independently of the presence of cyclic AMP and of the protein kinase C inhibitor H-7 and neither does the Ach effect on the Ca current seem to be correlated to a transient increase in intracellular Ca. Our results suggest that activation of the alpha-subunit of the PTX-sensitive G-protein could directly modulate the HVA Ca channel without involving second messenger systems.

Acetylcholine↗
PubMed1989

Activation of K+ currents in cultured Schwann cells is controlled by extracellular pH.

We analyzed the pH dependence of K+ currents recorded with the patch-clamp technique from cultured Schwann cells obtained from mouse dorsal root ganglia. Currents were activated at potentials more positive than -50 mV which was close to the resting membrane potential. Current amplitudes were affected by a change in extracellular pH (pHo), being increased at alkaline, and decreased at acidic pHo. The strongest effect of a pHo change was observed on currents activated close to the resting membrane potential suggesting a functional role for the pH sensitivity of K+ currents. Analysis of the time course of current activation at different pHo values led to the conclusion that the pH-sensitivity of K+ currents in Schwann cells is due to changes in surface charges shifting the potential sensed by the gating process of the channel. The reversal potential of the currents was not affected by a change in pHo. This observation and the finding that even a strong acidification to a pHo value of 5.0 did not lead to a blockade of the fully activated channel, indicate that the pH-sensitive charges are not located in the channel pore. Under the assumption that pHo changes in a peripheral nerve are associated with nerve activity as in the optic nerve, the pH-sensitive K+ channel in Schwann cells could serve to facilitate the spatial buffering of extracellular K+.

4-Aminopyridine↗
PubMed1989

GTP-binding proteins mediate acetylcholine inhibition of voltage dependent calcium channels in hippocampal neurons.

Acetylcholine (Ach) was tested for its effect on calcium currents in primary cultures of embryonic rat hippocampal neurones. 10 microM Ach reversibly depressed the high voltage activated (HVA) Ca current and the effect was blocked by atropine. When GTP binding proteins (G-proteins) were irreversibly activated by intracellular perfusing the neurons with GTP[S], one short-lasting application of Ach permanently inhibited Ca currents. Alternatively, the transmitter was inactive when the cells were preincubated with pertussis toxin, which inactivates some G-proteins by catalyzing ADP ribosylation. Our results suggest that a G-protein mediates the inhibitory modulation of the HVA Ca current by muscarinic agonists.

Acetylcholine↗
PubMed1989

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons.

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.

Animals↗
PubMed1989

Voltage-dependent and calcium-dependent inactivation of calcium channel current in identified snail neurones.

1. The dependence of Ca2+ current inactivation on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) was studied in TEA-loaded, identified Helix neurones which possess a single population of high-voltage-activated Ca2+ channels. During prolonged depolarization, the Ca2+ current declined from its peak with two clearly distinct phases. The time course of its decay was readily fitted by a double-exponential function. 2. In double-pulse experiments, the relationship between the magnitude of the Ca2+ current and the amount of Ca2+ inactivation was not linear, and considerable inactivation was present, even when conditioning pulses were to levels of depolarization so great that Ca2+ currents were near zero. Similar results were obtained when external Ca2+ was replaced by Ba2+. 3. In double-pulse experiments, hyperpolarization during the interpulse interval served to reprime a portion of the inactivated Ca2+ current for subsequent activation. The extent of repriming increased with hyperpolarization, reaching a maximum between -130 and -150 mV. The effectiveness of repriming hyperpolarizations was considerably increased when Ca2+ was replaced by Ba2+. 4. A significant fraction of inactivated Ca2+ channels can be recovered during hyperpolarizing pulses lasting only milliseconds. If hyperpolarizing pulses were applied before substantial inactivation of Ca2+ current, Ca2+ channels remained available for activation despite considerable Ca2+ entry. 5. The relationship between [Ca2+]i and inactivation was investigated by quantitatively injecting Ca2+-buffered solutions into the cells. The time course of Ca2+ current inactivation was unchanged at free [Ca2+] between 1 x 10(-7) and 1 x 10(-5) M. From 1 x 10(-7) to 1 x 10(-9) M, inactivation became progressively slower, mainly due to a decrease of the amplitude ratio (fast/slow) of the two components of inactivation, which fell from about unity to near zero at 1 x 10(-9) M. In double-pulse experiments, recovery from inactivation was enhanced in neurones that had been injected with Ca2+ chelator. 6. We conclude that inactivation of Ca2+ channels in these neurones depends on both [Ca2+]i and membrane potential. The voltage-dependent process may serve as a mechanism to quickly recover inactivated Ca2+ channels during repetitive firing despite considerable Ca2+ influx. 7. The results are discussed in the framework of a model which is based on two states of inactivation, INV and INCA, which represent different conformations of the inactivating substrate, and which are both reached from a lumped state of activation (A). Inactivation leads to high occupancy of INV during depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Distribution of calcium currents in sprouting PC12 cells.

Whole-cell calcium and barium currents were recorded from PC12 cell bodies and growth cones during nerve growth factor (NGF)-induced neurite outgrowth. Depolarizing voltage steps were applied to activate the currents, and pharmacological agents were used to separate them from other ionic currents. In recordings from growth cones still attached to neurites, current flow from central parts of the cell could be distinguished from the growth cone currents. On the other hand, currents from neuritic shafts and growth cones contributed to whole-cell currents recorded in the soma. Such currents were isolated by alternatively comparing recordings of whole-cell calcium currents with recordings in which calcium currents of defined parts of the cell were suppressed by exposing these regions to laminar streams of solutions with low calcium. The boundary between such streams and the bath solution was shown to be sharp using a calcium-selective microelectrode. The current deficits recorded when the growth cones were exposed to solutions with low calcium (growth cone currents) were within 10-50% of the total cell currents and similar to the currents recorded when the whole cell except the growth cone was exposed to low calcium. The current densities in the growth cones during initial sprouting were 5.4 times higher than those in the somata. Growth cone currents showed more inactivation than currents originating from the soma during pulses of 200 msec. In most experiments no calcium currents could be resolved in the neuritic shaft during initial neurite growth (1-10 d of NGF application), indicating low current density. In proximal segments of the neurites, however, a somatofugal decrease of the current density was observed. It is concluded from these results, that in outgrowing neurites of PC12 cells high densities of calcium channels are maintained in the growth cones, whereas in the neuritic shaft calcium channel density is initially low and later increases during consolidation of the neurites.

Animals↗
PubMed1988

Inactivation and block of calcium channels by photo-released Ca2+ in dorsal root ganglion neurons.

Calcium channels are inactivated by voltage and intracellular calcium. To study the kinetics and the mechanism of calcium-induced inactivation of calcium channels, a "caged" calcium compound, dimethoxy-nitrophen was used to photo-release about 50 microM calcium ion within 0.2 millisecond in dorsal root ganglion neurons. When divalent cations were the charge carriers, intracellular photo-release of calcium inactivated the calcium channel with an invariant rate [time constant (tau) approximately equal to 7 milliseconds]. When the monovalent cation sodium was the charge carrier, photorelease of calcium inside or outside of the cell blocked the channel rapidly (tau approximately equal to 0.4 millisecond), but the block was greater from the external side. Thus the kinetics of calcium-induced calcium channel inactivation depends on the valency of the permeant cation. The data imply that calcium channels exist in either of two conformational states, the calcium- and sodium-permeant forms, or, alternatively, calcium-induced inactivation occurs at a site closely associated with the internal permeating site.

Animals↗
PubMed1988

Similarity and mutual exclusion of NMDA- and proton-activated transient Na+-currents in rat tectal neurons.

Vertebrate neurons respond to rapid elevation of [H+]o with transient Na+-selective currents (INa(H]. Since INa(H) and voltage-activated Ca2+-currents (ICa(V] are mutually exclusive and similarly affected by inorganic and organic Ca2+-blockers, it has been suggested that such a Na+-permeable state evolves from protonation of Ca2+-channels. We show here that in cultured neurons from embryonic rat superior colliculus N-methyl-D-aspartate (NMDA) provides conditions for generation of a current identical with INa(H), but without the requirement of an increase in free [H+]o. The transient NMDA-activated current (I(NMDA)T) is occluded by INa(H). Its time course is similar to that of INa(H). Both currents are inactivated by long exposure to high [H+]o. I(NMDA)T displays a linear current-voltage (I-V) relationship under conditions which cause a negative slope in the I-V relationship of the persistent NMDA-activated current (I(NMDA)P). This suggests that the biphasic response of tectal neurons to the glutamate-agonist NMDA results from superposition of two different currents.

Animals↗
PubMed1988

Omega-conotoxin blockade distinguishes Ca from Na permeable states in neuronal calcium channels.

The blocking properties of the neurotoxic peptide omega-conotoxin GVIA (omega-CgTX), on neuronal Ca channels were investigated. In line with previous reports (Feldman et al. 1987; McCleskey et al. 1987), we found that micromolar concentrations of the toxin block selectively and persistently the high-threshold Ca channels of chick sensory neurons. The block by omega-CgTX could be partially relieved in low [Ca2+]o (less than 1 microM) toxin-free solutions, allowing Na ions to flow through open high-threshold Ca channels. Ca currents through these channels, however, remained permanently blocked on returning to normal Ca2+ toxin-free solutions. Also neurons which were preincubated with omega-CgTX in low Ca2+ (5 mM EGTA) failed to show high-threshold Ca currents during washing with normal Ca2+. Thus, appearance of Na currents through Ca channels in CgTX-pretreated cells was neither a consequence of unbinding of the toxin from its receptor site nor due to an interaction of EGTA with bound omega-CgTX. Na currents in CgTX-pretreated cells could be reversibly suppressed by bath applications of verapamil or by further addition of the toxin. At variance with Ca currents, block of Na currents by omega-CgTX was faster and reversible (KD 0.7 microM). Our data are consistent with the idea that neuronal Ca channels are in different conformational states when permeable to Ca2+ or Na+ ions and that omega-CgTX depresses persistently ion permeation primarily in the Ca-permeable state.

Animals↗
PubMed1988

Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurones.

1. In dissociated and cultured 1- to 2-day-old chick dorsal root ganglion cells, and in isolated outside-out membrane patches, a large transient current lasting 1-2 s could be activated upon step increases in [H+]o. The proton-induced current reversed direction at the Na+ equilibrium potential, was abolished completely in the absence of Na+, and was therefore labelled INa(H). 2. To investigate the activation and deactivation kinetics of INa(H) at the single-channel level, we employed isolated membrane patches and a method whereby we could change the external solution in less than 1 ms. 3. In outside-out membrane patches, INa(H) was fully activated within 2 ms between pH 6.7 and 5.7. Half-times of activation decreased with increasing [H+]o. The calculated association rate constant was 9.5 x 10(9) M-1 s-1. 4. Deactivation of INa(H), following a step reduction in [H+]o, occurred with half-times of within 1.3-2 ms. 5. In the continued presence of an activating solution (pH 6.7 and 1 mM-Ca2+), INa(H) inactivated slowly, with a time constant of about 300 ms. 6. Inactivation showed a limited dependence on [Ca2+]o. The time constant of inactivation increased from about 300-500 ms as [Ca2+]o was decreased from 5 to 0.1 mM. Further decrease in [Ca2+]o did not significantly increase the time course of inactivation. Increases in [Ca2+]i from 10(-9) to 10(-3) M had no effect on the activation or inactivation kinetics of INa(H). 7. Conditioning proton concentrations which by themselves failed to activate any channel openings, partially inactivated INa(H). 8. Recovery from inactivation appeared to follow a time course similar to that of inactivation itself. 9. INa(H) could not be activated in inside-out patches. A step increase in proton concentration outside a cell-attached patch was also ineffective at producing INa(H) in the patch. Intracellular pH between 7.9 and 6.7 had no effect on the activation or inactivation of INa(H). 10. The activation and inactivation kinetics were not significantly voltage dependent. 11. The single-channel conductance associated with the activation of INa(H) was 28 pS in symmetrical 120 mM-NaCl solutions and remained constant throughout the time course of INa(H). 12. During activation of the voltage-gated calcium current, ICa, a step increase in proton concentration caused a rapid (ca. 2 ms) suppression of ICa which was more than that predicted from the steady-state effects of H+ on ICa. This effect was independent of [Na+] and the direction of INa(H).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Studies on the development of voltage-activated calcium channels in vertebrate neurons.

The development of active Ca channels was studied in progenitors of chick dorsal root ganglion (DRG) cells. These were isolated using neuron and glia specific antibodies which killed differentiated cells. The low voltage activated, completely inactivating (LVA) Ca channel was found to be expressed first amongst channels carrying inward currents and thus providing substrates of excitability. The more ubiquitous high voltage activated and incompletely inactivating (HVA) Ca channel as well as the Tetrodotoxin-sensitive Na current appeared with a delay of about one half day. A similar time course of Ca-, but not Na-channel-expression was found in a parasympathetic preparation. Ca channels appear early and preferentially incorporated in growth zones of sprouting cells which may point to some function in the growth process. Incorporation is not directly promoted by nerve growth factor as shown in PC12 (pheochromocytoma) cells since it follows growth with considerable delay. LVA Ca channels are not expressed in this sympathetic cell line but in DRG cells they were observed to remain localized in the soma region. Such pattern in distribution was also concluded from results on rat hippocampal neurons and suggests different functional commitment of the Ca channel species.

Animals↗
PubMed1988

Development of inward currents in chick sensory and autonomic neuronal precursor cells in culture.

The development of ionic inward currents was studied in cultured neuronal precursors from chick sensory dorsal root ganglia (DRG) and compared with neuronal precursors from the cholinergic ciliary ganglia (CG) using whole cell patch-clamp recording. Neuronal precursors devoid of neuron-specific surface markers were isolated during the period of neuronal birth, i.e., at embryonic day (E) 6 from DRG and at E4.5 from CG. All neuronal precursor cells from DRG, as well as CG, showed outward K+ currents directly after they had attached to the substrate. During the first 5 hr in culture, half of the DRG cells had no inward currents at all, whereas the other half displayed a rapidly and fully inactivating Ca2+ current, which was activated with small depolarizing pulses from a holding potential of -80 mV to a -50 mV membrane potential (low-voltage-activated current, LVA). At these early stages, no other inward currents were resolved. TTX-blockable Na+ currents and slowly inactivating classical Ca2+ currents, which were activated with larger depolarizing pulses to a -20 mV membrane potential (high-voltage-activated currents, HVA) appeared concurrently after 15-20 hr in culture. In contrast, more than half of the CG cells showed LVA currents, as well as Na+ currents, as early as during the first 5 hr in culture. The HVA Ca2+ currents from the majority of the cells could be recorded only after 10-15 hr in culture. In both types of precursor-derived neurons, the LVA Ca2+ current preceded the classical HVA Ca2+ current. However, the temporal relation of the first Na+ currents to the first HVA Ca2+ currents seemed to be different in the 2 preparations. In DRG cells, Na+ and HVA Ca2+ currents appeared at the same time, whereas in CG cells, the HVA Ca2+ current showed a time lag with respect to the Na+ current. In addition, the relative amplitudes of the currents differed in the CG and DRG cells. This shows that as early as E4-6, shortly after their terminal mitosis, neurons from distinct peripheral ganglia in chick vary in the development of their basic ionic currents.

Animals↗
PubMed1987

Glutamate-induced ionic currents in cultured neurons from the rat superior colliculus.

The ionic currents induced in cultured rat superior colliculus neurons by rapid application of glutamate (Glut) and the glutamate receptor agonists quisqualate (Quis) and N-methyl-D-aspartate (NMDA) were examined using the whole-cell patch clamp technique. Dissociated cell cultures consisting exclusively of superficial gray layer neurons from rats aged E21-P2 were used. After 7-10 days in vitro, all neurons responded to Glut and the selective agonists, NMDA and Quis. Glut was a mixed agonist, and a variable fraction (10-100%) of Glut-activated currents was due to involvement of NMDA receptors. The NMDA response was strongly regulated by extracellular Ca and Mg levels and modified by exposure to Quis. Quis transiently removed the block of NMDA-activated currents by D-amino-phosphonovaleric acid (APV).

Animals↗