Developmental regulation of potassium channels and the impact on neuronal differentiation.
Explore the source record for details and available documents.
Action Potentials↗
Biomedical subjects
N C Spitzer
Publications and source records attributed to N C Spitzer.
Reconstruction of action potential development from whole-cell currents of differentiating spinal neurons.
The duration and ionic dependence of action potentials are developmentally regulated. Voltage-clamp recordings of amphibian spinal neurons have revealed alterations in five currents. To determine whether the changes in the currents are sufficient to produce the change in action potential duration and ionic dependence, we constructed a Hodgkin-Huxley model of electrical excitability of these neurons. The model shows that the equations describing the voltage-clamped currents of young and mature neurons generate action potentials appropriate in duration and ionic dependence for each developmental stage. Moreover, the observed changes in the currents are quantitatively sufficient to produce the changes in the action potential. The effect of the change in each current is detectable in the model. However, the increase in amplitude of the delayed-rectifier potassium current has the largest effect. The model further shows that changes in action potential duration could be achieved with changes in kinetics rather than amplitude of this current, or with changes in amplitudes of other currents. Thus, although increase in amplitude of the delayed rectifier plays a pivotal role in the maturation of excitability, it is not uniquely positioned to govern the action potential duration.
Action Potentials↗
A developmental handshake: neuronal control of ionic currents and their control of neuronal differentiation.
Explore the source record for details and available documents.
Action Potentials↗
Differentiation of delayed rectifier potassium current in embryonic amphibian myocytes.
The developmentally regulated expression of prolonged outward potassium currents influences the extent to which sustained inward currents contribute to the action potential at early stages of differentiation. In amphibian spinal neurons, the long duration and calcium dependence of the embryonic action potential and the amount of calcium influx are largely determined by the extent of maturation of the delayed rectifier potassium current (IKv). We have undertaken a parallel study of differentiation of myocytes, in which action potentials are brief and sodium-dependent even at early stages. The early expression of electrical excitability in embryonic amphibian myocytes growing in culture has been examined previously using intracellular voltage recording techniques. The membrane exhibits a delayed rectification in response to depolarization at times earlier than those at which impulses can first be generated. We have examined the differentiation of this outward current in embryonic myocytes developing in vitro, using whole cell voltage clamp. IKv is initially absent. When first recorded it is small and slowly activating but undergoes sixfold increases in both density and rate of activation during the first day in culture. This maturation is dependent upon transcription, and both rate and density are influenced by the presence of other cell types. The large amplitude of the outward delayed rectifier prevents expression of long duration action potentials.
Animals↗
Calcium-induced release of calcium regulates differentiation of cultured spinal neurons.
Voltage-dependent calcium influx has been shown to regulate the differentiation of cultured amphibian spinal neurons. We have examined the transient elevation of intracellular calcium induced by depolarization, using calcium indicators and confocal microscopy with high temporal and spatial resolution. Rapid calcium elevations in both the nucleus and the cytosol are primarily due to calcium-dependent release of calcium from intracellular stores. Depletion of stores associated with the endoplasmic reticulum reduces all transients. Elevations diminish with neuronal maturation. Depletion of stores of intracellular calcium at early times affects neuronal differentiation in a manner similar to the prevention of influx. The results indicate that both influx and release are necessary to promote neuronal differentiation.
Animals↗
Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons.
The delayed rectifier current of embryonic Xenopus spinal neurons plays the central role in developmental conversion of calcium-dependent action potentials to sodium-dependent spikes. During its maturation, this potassium current undergoes a pronounced increase in rate of activation. The mechanism underlying the change in kinetics was analyzed with whole-cell voltage clamp of neurons cultured under various conditions. Calcium is necessary at an early stage of development, to permit influx that triggers subsequent release of calcium from intracellular stores. Its action is prevented by depletion of protein kinase C and mimicked by stimulation of the kinase. Calcium influx through voltage-dependent channels at early stages of development regulates the differentiation of potassium current kinetics and modulation of the ionic dependence of action potentials.
Animals↗
Spontaneous calcium influx and its roles in differentiation of spinal neurons in culture.
Stimulation of embryonic amphibian spinal neurons has been shown to produce calcium-dependent action potentials of long duration at early stages of development. These impulses become brief and sodium-dependent upon further differentiation. The neurons are now shown to exhibit spontaneous, transient elevations of intracellular calcium in culture during the early developmental period when activity produces greatest calcium influx. Removal of extracellular calcium during this period alone is sufficient to perturb differentiation, and influx through voltage-dependent calcium channels is shown to be required for standard development of neuronal phenotypes. No large changes in steady-state calcium levels occur in the cytoplasm during the maturation of cultured neurons despite a reduction of the calcium-dependent component of the impulse. Transient elevation of intracellular calcium is necessary for standard cytodifferentiation and may provide a link between electrical activity and gene expression.
Action Potentials↗
Differentiation of IKA in amphibian spinal neurons.
We have examined the development of an inactivating outward current in embryonic amphibian neurons differentiating in culture. On the basis of ionic selectivity, voltage dependence of activation and inactivation and pharmacological sensitivity, it is similar to A currents described in other neurons. In these embryonic neurons the A current appears later than other voltage-dependent currents studied previously. Furthermore, its maturation extends to times later than those required for the differentiation of another potassium current, the voltage-dependent delayed rectifier. These changes in A current are consistent with parallel changes in the action potential and excitability of the developing neurons.
Action Potentials↗
A critical period of transcription required for differentiation of the action potential of spinal neurons.
The early development of excitability of amphibian spinal neurons is characterized by a change from a long Ca2(+)-dependent action potential to a brief Na(+)-dependent impulse. The delayed rectifier K+ current plays a major role in this cell autonomous differentiation. Here we show that the maturation of the delayed rectifier current, and hence the action potential, involves a critical period of mRNA synthesis. It is blocked by inhibition of transcription during an early period of development in culture and fails to develop following removal of the inhibitor and resumption of RNA synthesis. However, the development of an inactivating K+ A-current recovers in these neurons, indicating that some programs of neuronal development are affected during this critical period, while others are spared.
Action Potentials↗
Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation.
Single-channel K+ currents were recorded from young and mature spinal neurons cultured from Xenopus embryos to examine the bases of the developmental increases in density and in rate of activation of the macroscopic voltage-dependent delayed rectifier K+ current (IKv). K+ channels of three conductance classes (integral of 80, 30, and 15 pS) are present at both ages, but only the intermediate and small conductance classes are voltage-dependent and thus underlie IKv. The increase in the density of IKv is due to increases in the numbers of intermediate and small channels per cell, but not to changes in their open probabilities. The increase in rate of activation of IKv results from a change in the activation kinetics of the intermediate channel class alone.
Animals↗
Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons.
Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.
Action Potentials↗
Both barium and calcium activate neuronal potassium currents.
Amphibian spinal neurons in culture possess both rapidly inactivating and sustained calcium-dependent potassium current components, similar to those described for other cells. Divalent cation-dependent whole-cell outward currents were isolated by subtracting the voltage-dependent potassium currents recorded from Xenopus laevis neurons in the presence of impermeant cadmium (100-500 microM) from the currents produced without cadmium but in the presence of permeant divalent cations (50-100 microM). These concentrations of permeant ions were low enough to avoid contamination by macroscopic inward currents through calcium channels. Calcium-dependent potassium currents were reduced by 1 microM tetraethylammonium. These currents can also be activated by barium or strontium. Barium as well as calcium activated outward currents in young neurons (6-8 hr) and in relatively mature neurons (19-26 hr in vitro). However, barium influx appeared to suppress the sustained voltage-dependent potassium current in most cells. Barium also activated at least one class of potassium channels observed in excised membrane patches, while blocking others. The blocking action may have masked and hindered detection of the stimulatory action of barium in other systems.
Animals↗
Autonomous early differentiation of neurons and muscle cells in single cell cultures.
The extent to which early differentiation of neurons and muscle cells is autonomous or governed by soluble factors released from other cells has been examined by following development of single cells plated alone in a simple, defined culture medium. The differentiation of electrical excitability and sensitivity to neurotransmitters of amphibian spinal neurons and trunk muscle in Xenopus embryos has already been described. For both cell types, differentiation in cultures containing relatively large numbers of dispersed cells parallels development in vivo, with respect to qualitative changes in membrane properties and the time course of development. Cell contacts are not required for this process. Here we show that the differentiation of membrane properties of single, isolated cells exhibits a similar set of changes, although muscle cells develop more slowly in some respects and all cells survive for a shorter period of time. The results suggest that the continued presence of specific extracellular differentiation-promoting factors is not required for these early steps of neuronal development, although a role for such factors in development of myocytes cannot be excluded. In contrast, survival factors secreted by other cells may be necessary to prolong the lifetimes of dissociated cells.
Acetylcholine↗
The changing view of neural specificity.
The generation of specific patterns of neuronal connections has usually been regarded as a central problem in neurobiology. The prevailing view for many years has been that these connections are established by complementary recognition molecules on the pre- and postsynaptic cells (the chemoaffinity theory). Experimental results obtained in the past decade, however, indicate that the view that axon guidance and synaptogenesis proceed according to restrictive chemical markers is too narrow. Although a more rigid plan may prevail in some invertebrates, the formation of specific connections in vertebrates also involves competition between axon terminals, trophic feedback between pre- and postsynaptic cells, and modification of connections by functional activity.
Animals↗
Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx.
The development of the action potential and responses to neurotransmitters have been described for a population of embryonic spinal neurons developing in vivo. A comparable pattern is seen for spinal neurons developing in dissociated cell culture. The impulse appears very early in this developmental sequence, and the action potential involves a large inward Ca2+ current. Since Ca2+ is a ubiquitous intracellular regulator, we questioned whether a large influx of Ca2+ is necessary for the subsequent differentiation of membrane properties. Embryonic Xenopus neurons grown in normal culture medium do not make Ca2+- or Na+-dependent action potentials in their cell bodies in a Ca2+-free saline containing tetrodotoxin (TTX). To achieve a chronic blockade of impulse activity, neurons were grown in a medium in which Ca2+ was replaced by Mg2+, and to which 1 mM EGTA was added. In some instances TTX was present. Neurons grown in these experimental culture media extend neurites more rapidly than controls. Action potentials cannot be elicited from neurons when examined in experimental medium. However, examination in saline reveals that the change in the ionic dependence of the impulse is indistinguishable from that observed in neurons grown in normal medium. Furthermore, the time of onset of responses to GABA is unaffected by this experimental treatment. Thus the expression of Ca2+- and Na+-dependent action potentials seems not to play a part in the early differentiation of these membrane properties. However, the later development of GABA sensitivity is reduced.
Action Potentials↗
The appearance and development of neurotransmitter sensitivity in Xenopus embryonic spinal neurones in vitro.
We have determined the time of onset and examined some of the properties of neurotransmitter sensitivity in Xenopus spinal neurones developing in dissociated cell culture. These cells are initially insensitive, but acquire responses to several agonists over a period of 6 h. Nearly one-third of the neurones were depolarized by gamma-aminobutyric acid (GABA) or by both GABA and glycine; these cells were not affected by glutamate. The reversal potential of the ionophoretic GABA response is -35 mV. These neurones are likely to be Rohon-Beard neurones. Roughly two-thirds of the neurones were depolarized by glutamate and hyperpolarized by GABA and by glycine. The reversal potential of the ionophoretic GABA response is -58 mV. These neurones are likely to include motoneurones. A quantitative measure of the sensitivity to a given GABA dose was obtained at early and intermediate stages of development. The mean 'sensitivity index' (ionophoretic sensitivity/input resistance) for both classes of neurones in vitro was initially the same as that seen in Rohon-Beard neurones in vivo. This sensitivity index did not increase with time in culture to attain the value at intermediate stages in vivo. The development of chemosensitivity in Rohon-Beard-like neurones in these cultures resembles that of Rohon-Beard neurones in the spinal cord with respect to the time of onset of responses to GABA, the reversal potential, pharmacology and desensitization of these responses, and the spectrum of agonists to which they are sensitive. It differs in the absence of a developmental increase in sensitivity to GABA. The development of chemosensitivity in motoneurone-like neurones in these cultures parallels that of Rohon-Beard-like neurones, with respect to the time of onset and level of sensitivity, as well as susceptibility to pharmacological blockers. Several features of normal neurotransmitter sensitivity, like features of the action potential, differentiate in culture in the absence of normal cellular interactions.
Action Potentials↗
The absence of calcium blocks impulse-evoked release of acetylcholine but not de novo formation of functional neuromuscular synaptic contacts in culture.
We have examined the role of calcium-dependent, impulse-evoked release of acetylcholine (ACh) in the formation of functional neuromuscular contacts. Dissociated cell cultures prepared from Xenopus laevis embryos were maintained either in standard medium containing calcium or in calcium-free medium. Both the number of neuron-myocyte contacts and the number of terminations of neurites on myocytes were reduced in Ca-free medium. Intracellular recordings from neuron-myocyte pairs during perfusion with a standard saline revealed that functional synaptic contacts were formed in Ca-free medium, although with a reduced frequency (approximately 30% of controls). Postsynaptic potentials elicited by neuronal action potentials were smaller than those observed after growth in standard medium. The frequency of small, spontaneously occurring potentials was reduced by a factor of 10. Ca-free saline prevented the impulse-evoked release of transmitter from neurons in Ca-free and control cultures. Labeling with alpha-bungarotoxin and iontophoretic application of ACh revealed no ACh receptor clusters in the membrane of myocytes grown in Ca-free medium. Our results suggest that evoked, vesicular release is not required for the initial formation of neuromuscular contacts, although it may be involved in further maturation of synapses. Roles for spontaneous quantal or non-quantal release have not been excluded.
Acetylcholine↗
Reassembly of protein-lipid complexes into large bilayer vesicles: perspectives for membrane reconstitution.
Protein-lipid complexes in apolar solvents reassemble into large bilayer protein-lipid vesicles (PLVs) with diameters of several micrometers. PLVs form spontaneously upon hydration of the protein-lipid complex residue after solvent removal. This procedure has been applied to the following membrane proteins: bovine and squid rhodopsin, reaction centers from Rhodopseudomonas sphaeroides, beef heart cytochrome c oxidase, and acetylcholine receptors from Torpedo californica. PLVs have a large internal aqueous space (e.g., 790 mul/mg of lipid for cattle rhodopsin vesicles). Freeze-fracture replicas of PLVs revealed that both internal and external leaflets contained numerous intramembranous particles with diameters between 80 and 120 A, depending on the specific protein incorporated in the membrane. The optical spectral properties of rhodopsin and reaction centers in PLVs were similar to those recorded in the respective natural membrane. Furthermore, bovine rhodopsin in PLVs was chemically regenerable with 9-cis-retinal. Actinic illumination induced proton efflux from reaction center vesicles that was abolished by proton ionophores. Therefore, this method is suitable for the incorporation of some membrane proteins in their functional state. PLVs were penetrated with microelectrodes and visualized by the injection of a fluorescent dye. Preliminary electrical recordings were obtained by sealing PLVs to a hole in a septum separating two aqueous compartments. These studies suggest that PLVs assembled by this procedure permit the simultaneous analysis of reconstituted membranes by chemical, optical, and electrical techniques.
Animals↗