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At least 19 recordsLinked to original sources
PubMed1992

Inhibition of protein kinases in rat pheochromocytoma (PC12) cells promotes morphological differentiation and down-regulates ion channel expression.

We have studied morphological differentiation and ion channel expression in PC12 cells under different culture conditions. Differentiation mediated by nerve growth factor (NGF) was compared with that induced by depletion and inhibition of protein kinases (phorbol ester beta-PMA plus staurosporine). Morphological differentiation was similar under both conditions. However, ion channel densities, studied by means of the patch-clamp technique, were enhanced by NGF and reduced by beta-PMA+staurosporine. Similar changes were also observed for omega-conotoxin-sensitive Ca2+ channels by measuring radioligand binding. The decrease in Ca2+ channel density, after treatment of the cells with beta-PMA+staurosporine, resulted in a reduced increase in the intracellular Ca2+ concentration during K+ depolarization. We conclude that morphological differentiation, but not ion channel expression, can occur during depression of protein kinase activities in PC12 cells.

Alkaloids↗
PubMed1992

Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.

Animals↗
PubMed1992

Astrocytes, as well as neurons, express a diversity of ion channels.

The electrophysiologist's view of brain astrocytes has changed markedly in recent years. In the past astrocytes were viewed as passive, K+ selective cells, but it is now evident that they are capable of expressing voltage- and ligand-activated channels previously thought to be restricted to neurons. The functional importance of most of these ion channels is not understood at present. However, from studies of astrocytes cultured from different species and brain regions, we learned that like their neuronal counterparts astrocytes are a heterogeneous group of brain cells showing similar heterogeneity in their ion-channel expression. Not only are subpopulations of astrocytes within areas of the brain equipped with specific sets of ion channels but, furthermore, regional heterogeneity is apparent. In addition, astrocyte ion channel expression is dynamic and changes during development. Some ion channels are only expressed postnatally, yet others appear to be expressed only during certain stages of development. Interestingly, the expression of some astrocyte channels, including Na+, Ca2+, and some K+ channels, appears to be controlled by neurons via mechanisms that are presently unknown. Some studies suggest roles for astrocyte channels in basic cell processes such as cell proliferation. Thus, although the role of some astrocyte channels remains unclear, our understanding of astrocyte physiology is starting to take shape and points towards roles of ion channels not involved in electrogenesis.

Animals↗
PubMed1992

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.

Animals↗
PubMed1992

Cloning and functional expression of a cDNA encoding the mouse beta 2 subunit of the kainate-selective glutamate receptor channel.

The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

Amino Acid Sequence↗
PubMed1992

New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.

Amino Acid Sequence↗
PubMed1992

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.

Amino Acid Sequence↗
PubMed1992

Electrophysiological properties of a cell line of the gonadotrope lineage.

The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, alpha T3-1, has been generated that makes and secretes the alpha-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in alpha T3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium channels with rapid kinetics, several types of potassium channels, including Ca2(+)-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13-acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH.

Animals↗
PubMed1992

The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits.

A new ionotropic glutamate receptor subunit termed KA-2, cloned from rat brain cDNA, exhibits high affinity for [3H]kainate (KD approximately 15 nM). KA-2 mRNA is widely expressed in embryonic and adult brain. Homomeric KA-2 expression does not generate agonist-sensitive channels, but currents are observed when KA-2 is coexpressed with GluR5 or GluR6 subunits. Specifically, coexpression of GluR5(R) and KA-2 produces channel activity, whereas homomeric expression of either subunit does not. Currents through heteromeric GluR5(Q)/KA-2 channels show more rapid desensitization and different current-voltage relations when compared with GluR5(Q) currents. GluR6/KA-2 channels are gated by AMPA, which fails to gate homomeric GluR6 receptor channels. These results suggest possible in vivo partnership relations for high affinity kainate receptors.

Amino Acid Sequence↗
PubMed1992

Functional expression of an inactivating potassium channel cloned from human heart.

Recently a putative K+ channel with homology to the Shaker family of potassium channels has been cloned from human ventricular myocardium. However, proof that the cDNA encodes a K+ channel requires appropriate translation and expression of a functional ion-selective channel. Therefore, expression of this putative human K+ channel DNA was attempted by cytoplasmic injections of in vitro transcribed cRNA into Xenopus laevis oocytes and screening by two-electrode voltage-clamp methods. This resulted in expression of voltage-gated channels that rapidly inactivated (time constant of inactivation, 47.6 +/- 3.6 msec; 0 mV; n = 10) and were at least 50 times more selective for K+ than Na+ (Na+/K+ permeability ratio of 0.02). The channels showed voltage-dependent activation (half-maximal voltage, -34 +/- 0.7 mV; n = 5), and 50% of the channels were inactivated within 2 seconds when the membrane potential was clamped near -60 mV (half-maximal voltage, -62 +/- 7 mV; n = 10). The expressed protein resulted in a K+ current that had many properties similar to the 4-aminopyridine-sensitive calcium-insensitive component of the cardiac transient outward current that is observed in native cardiac myocytes and thus may serve as one molecular substrate for this current.

Animals↗
PubMed1992

Acetylcholine receptor channel structure probed in cysteine-substitution mutants.

In order to understand the structural bases of ion conduction, ion selectivity, and gating in the nicotinic acetylcholine receptor, mutagenesis and covalent modification were combined to identify the amino acid residues that line the channel. The side chains of alternate residues--Ser248, Leu250, Ser252, and Thr254--in M2, a membrane-spanning segment of the alpha subunit, are exposed in the closed channel. Thus alpha 248-254 probably forms a beta strand, and the gate is closer to the cytoplasmic end of the channel than any of these residues. On channel opening, Leu251 is also exposed. These results lead to a revised view of the closed and open channel structures.

Acetylcholine↗
PubMed1992

Cloning and expression of the epsilon 4 subunit of the NMDA receptor channel.

The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.

Amino Acid Sequence↗
PubMed1992

Molecular diversity of the NMDA receptor channel.

Two novel subunits of the mouse NMDA receptor channel, the epsilon 2 and epsilon 3 subunits, have been identified by cloning and expression of complementary DNAs. The heteromeric epsilon 1/zeta 1, epsilon 2/zeta 1 and epsilon 3/zeta 1 NMDA receptor channels exhibit distinct functional properties in affinities for agonists and sensitivities to competitive antagonists and Mg2+ block. In contrast to the wide distribution of the epsilon 1 and zeta 1 subunit messenger RNAs in the brain, the epsilon 2 subunit mRNA is expressed only in the forebrain and the epsilon 3 subunit mRNA is found predominantly in the cerebellum. The epsilon 1/zeta 1 and epsilon 2/zeta 1 channels expressed in Xenopus oocytes, but not the epsilon 3/zeta 1 channel, are activated by treatment with 12-O-tetradecanoylphorbol 13-acetate. These findings suggest that the molecular diversity of the epsilon subunit family underlies the functional heterogeneity of the NMDA receptor channel.

Amino Acid Sequence↗
PubMed1992

The third gamma subunit of the gamma-aminobutyric acid type A receptor family.

Cloned cDNAs encoding a member of the gamma-aminobutyric acid type A receptor gamma-subunit class were isolated from rat-brain-mRNA-derived libraries. The gamma 3 mRNA is present in cortex, claustrum, caudate putamen, and some thalamic nuclei, particularly the medial geniculate nucleus, where it is the predominant gamma-subunit transcript. The gamma 3 gene is expressed at very low levels in cerebellum and hippocampus. In coexpression experiments with the alpha 1 and beta 2 subunits, gamma 3 imparts benzodiazepine binding to gamma-aminobutyric acid type A receptors and forms gamma-aminobutyric acid-gated benzodiazepine-modulated chloride channels that exhibit a larger conductance than alpha 1 beta 2 receptor channels. Furthermore, the presence of gamma 3 in place of gamma 2 in alpha 1 beta 2 gamma x receptors generates a marked decrease in the affinity of agonists while leaving the affinity of antagonists or negative modulators largely unaffected.

Amino Acid Sequence↗
PubMed1992

Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist.

Primary cultures of precursor cells from mouse and rat brown adipose tissue (BAT) were used to study the effect of a new beta-agonist (ICI D7114) on the uncoupling protein (UCP) gene expression. ICI 215001 (the active metabolite of D7114) increased the expression of UCP and its mRNA in brown adipocytes differentiating in vitro in a dose-dependent manner. This stimulating effect was not inhibited by propranolol, a non-specific beta-antagonist, but was partially reduced by bupranolol, a beta 3-antagonist. No expression of UCP mRNA was ever induced by ICI 215001 in white adipocytes differentiated in vitro. It was concluded that the drug could affect the brown adipose cells through a beta 3-pathway. It could clearly modulate the expression of UCP in brown adipocytes differentiated in vitro, but was not able by itself to turn on the gene.

Adipose Tissue, Brown↗