External calcium ions are required for potassium channel gating in squid neurons

The effects of calcium removal on the voltage-dependent potassium channels of isolated squid neurons were studied with whole cell patch-clamp techniques. When the calcium ion concentration was lowered from 10 to 0 millimolar (that is, no added calcium), potassium channel activity, identified from its characteristic time course, disappeared within a few seconds and there was a parallel increase in resting membrane conductance and in the holding current. The close temporal correlation of the changes in the three parameters suggests that potassium channels lose their ability to close in the absence of calcium and simultaneously lose their selectivity. If potassium channels were blocked by barium ion before calcium ion was removed, the increases in membrane conductance and holding current were delayed or prevented. Thus calcium is an essential cofactor in the gating of potassium channels in squid neurons.     

Protective role of ATP-sensitive potassium channels in hypoxia-induced generalized seizure

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels are activated by various metabolic stresses, including hypoxia. The substantia nigra pars reticulata (SNr), the area with the highest expression of K(ATP) channels in the brain, plays a pivotal role in the control of seizures. Mutant mice lacking the Kir6.2 subunit of K(ATP) channels [knockout (KO) mice] were susceptible to generalized seizures after brief hypoxia. In normal mice, SNr neuron activity was inactivated during hypoxia by the opening of the postsynaptic K(ATP) channels, whereas in KO mice, the activity of these neurons was enhanced. K(ATP) channels exert a depressant effect on SNr neuronal activity during hypoxia and may be involved in the nigral protection mechanism against generalized seizures.     

Stretch-inactivated ion channels coexist with stretch-activated ion channels

Stretch-activated ion channels of animal, plant, bacterial, and fungal cells are implicated in mechanotransduction and osmoregulation. A new class of channel has now been described that is stretch-inactivated. These channels occur in neurons, where they coexist with stretch-activated channels. Both channels are potassium selective. The differing stretch sensitivities of the two channels minimize potassium conductance over an intermediate range of tension, with the consequence that, over this same range, voltage-gated calcium channels are most readily opened. Thus, by setting the relation between membrane tension and transmembrane calcium fluxes, stretch-sensitive potassium channels may participate in the control of calcium-dependent motility in differentiating, regenerating, or migrating neurons.     

A distinct potassium channel polypeptide encoded by the Drosophila eag locus.

Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether à go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.     

Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk

The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.     

Immunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission

Immunohistochemical analysis of adenosine deaminase in rat brain revealed an extensive plexus of adenosine deaminase-containing neurons in the basal hypothalamus. These neurons converged on and were most numerous in three major centers, namely, the tuberal, caudal, and postmammillary caudal magnocellular nuclei. Most other brain regions were devoid of cells containing adenosine deaminase. Some adenosine deaminase-containing neurons were retrogradely labeled with the fluorescent dye fast blue when the dye was injected into the frontal cortex and striatum. Specific populations of neurons having high levels of adenosine deaminase may release adenosine as a neurotransmitter.     

A targeting motif involved in sodium channel clustering at the axonal initial segment

The sorting of sodium channels to axons and the formation of clusters are of primary importance for neuronal electrogenesis. Here, we showed that the cytoplasmic loop connecting domains II and III of the Nav1 subunit contains a determinant conferring compartmentalization in the axonal initial segment of rat hippocampal neurons. Expression of a soluble Nav1.2II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, this motif may play a fundamental role in controlling electrical excitability during development and plasticity.     

A G protein gamma subunit shares homology with ras proteins

Guanine nucleotide binding proteins (G proteins) that transduce signals from cell surface receptors to effector molecules are made up of three subunits, alpha, beta, and gamma. A complementary DNA clone that encodes a 71-amino acid protein was isolated from bovine brain; this protein contains peptide sequences that were derived from the purified gamma subunit of Gi and Go. The primary sequence of this G protein gamma subunit (G gamma) has 55 percent homology to the gamma subunit of transducin (T gamma) and also has homology to functional domains of mammalian ras proteins. The probe for isolating the clone was generated with the use of the polymerase chain reaction (PCR). The extent of divergence between T gamma and G gamma, the isolation of homologous PCR-generated fragments, and the differences between the predicted amino acid sequence of G gamma and that derived from the gamma subunit of Gi and Go indicate that gamma subunits are encoded by a family of genes.     

Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels

Voltage-dependent ion channels are responsible for electrical signaling in neurons and other cells. The main classes of voltage-dependent channels (sodium-, calcium-, and potassium-selective channels) have closely related molecular structures. For one member of this superfamily, the transiently voltage-activated Shaker H4 potassium channel, specific amino acid residues have now been identified that affect channel blockade by the small ion tetraethylammonium, as well as the conduction of ions through the pore. Furthermore, variation at one of these amino acid positions among naturally occurring potassium channels may account for most of their differences in sensitivity to tetraethylammonium.     

Synaptic assembly of the brain in the absence of neurotransmitter secretion

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.     

A G protein couples serotonin and GABAB receptors to the same channels in hippocampus

Both serotonin and the selective gamma-aminobutyric acidB (GABAB) agonist, baclofen, increase potassium (K+) conductance in hippocampal pyramidal cells. Although these agonists act on separate receptors, the potassium currents evoked by the agonists are not additive, indicating that the two receptors share the same potassium channels. Experiments with hydrolysis-resistant guanosine triphosphate (GTP) and guanosine diphosphate analogs and pertussis toxin indicate that the opening of the potassium channels by serotonin and GABAB receptors involves a pertussis toxin-sensitive GTP-binding (G) protein, which may directly couple the two receptors to the potassium channel.     

Functional properties of rat brain sodium channels expressed in a somatic cell line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Expression of a cloned rat brain potassium channel in Xenopus oocytes

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.     

NMDA receptor-mediated K+ efflux and neuronal apoptosis

Neuronal death induced by activating N-methyl-D-aspartate (NMDA) receptors has been linked to Ca2+ and Na+ influx through associated channels. Whole-cell recording from cultured mouse cortical neurons revealed a NMDA-evoked outward current, INMDA-K, carried by K+ efflux at membrane potentials positive to -86 millivolts. Cortical neurons exposed to NMDA in medium containing reduced Na+ and Ca2+ (as found in ischemic brain tissue) lost substantial intracellular K+ and underwent apoptosis. Both K+ loss and apoptosis were attenuated by increasing extracellular K+, even when voltage-gated Ca2+ channels were blocked. Thus NMDA receptor-mediated K+ efflux may contribute to neuronal apoptosis after brain ischemia.     

Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels

Sulfonylurea-sensitive adenosine triphosphate (ATP)-regulated potassium (KATP) channels are present in brain cells and play a role in neurosecretion at nerve terminals. KATP channels in substantia nigra, a brain region that shows high sulfonylurea binding, are inactivated by high glucose concentrations and by antidiabetic sulfonylureas and are activated by ATP depletion and anoxia. KATP channel inhibition leads to activation of gamma-aminobutyric acid (GABA) release, whereas KATP channel activation leads to inhibition of GABA release. These channels may be involved in the response of the brain to hyper- and hypoglycemia (in diabetes) and ischemia or anoxia.     

Mechanism of the rapid effect of 17 beta-estradiol on medial amygdala neurons

The mechanism by which sex steroids rapidly modulate the excitability of neurons was investigated by intracellular recording of neurons in rat medial amygdala brain slices. Brief hyperpolarization and increased potassium conductance were produced by 17 beta-estradiol. This effect persisted after elimination of synaptic input and after suppression of protein synthesis. Thus, 17 beta-estradiol directly changes the ionic conductance of the postsynaptic membrane of medial amygdala neurons. In addition, a greater proportion of the neurons from females than from males responded to 17 beta-estradiol.     

Brain region and gene specificity of neuropeptide gene expression in cultured astrocytes

Astrocytes have many neuronal characteristics, such as neurotransmitter receptors, ion channels, and neurotransmitter uptake systems. Cultured astrocytes were shown to express certain neuropeptide genes, with specificity for both the gene expressed and the brain region from which the cells were prepared. Somatostatin messenger RNA and peptides were detected only in cerebellar astrocytes, whereas proenkephalin messenger RNA and enkephalin peptides were present in astrocytes of cortex, cerebellum, and striatum. Cholecystokinin was not expressed in any of the cells. These results support the hypothesis that peptides synthesized in astrocytes may play a role in the development of the central nervous system.     

Long-term modulation of electrical synapses in the mammalian thalamus

Electrical synapses are common between inhibitory neurons in the mammalian thalamus and neocortex. Synaptic modulation, which allows flexibility of communication between neurons, has been studied extensively at chemical synapses, but modulation of electrical synapses in the mammalian brain has barely been examined. We found that the activation of metabotropic glutamate receptors, via endogenous neurotransmitter or by agonist, causes long-term reduction of electrical synapse strength between the inhibitory neurons of the rat thalamic reticular nucleus.     

Role of ER export signals in controlling surface potassium channel numbers

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.