The human NTERA2 neural cell line generates neurons on growth under neural stem cell conditions and exhibits characteristics of radial glial cells.

NTERA2 cells are a human neural cell line generating neurons after exposure to retinoic acid and, as such, are widely used as a model of neurogenesis. We report that these cells form spheres when grown in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). These spheres were found to express markers of radial glial cells such as, Pax6, glutamate transporter (GLAST), tenascin C, brain lipid-binding protein (BLBP), and the 3CB2 antigen. On plating on an adhesive substrate, NTERA2 spheres generate a large percentage of immature neurons (30-50%) together with a minority of cells of the oligodendrocyte lineage. Thus NTERA2 cells share properties with neural stem cells. However, at variance with the latter, we found that they produce their own bFGF implicated in an autocrine or paracrine proliferative loop and that they do not generate astrocytes after differentiation. These results provide an interesting model to study radial glial cells and their role in human neurogenesis.

Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors.

Nonsteroid anti-inflammatory drugs (NSAIDs) are major drugs against inflammation and pain. They are well known inhibitors of cyclooxygenases (COXs). However, many studies indicate that they may also act on other targets. Acidosis is observed in inflammatory conditions such as chronic joint inflammation, in tumors and after ischemia, and greatly contributes to pain and hyperalgesia. Administration of NSAIDs reduces low-pH-induced pain. The acid sensitivity of nociceptors is associated with activation of H(+)-gated ion channels. Several of these, cloned recently, correspond to the acid-sensing ion channels (ASICs) and others to the vanilloid receptor family. This paper shows (1) that ASIC mRNAs are present in many small sensory neurons along with substance P and isolectin B4 and that, in case of inflammation, ASIC1a appears in some larger Abeta fibers, (2) that NSAIDs prevent the large increase of ASIC expression in sensory neurons induced by inflammation, and (3) that NSAIDs such as aspirin, diclofenac, and flurbiprofen directly inhibit ASIC currents on sensory neurons and when cloned ASICs are heterologously expressed. These results suggest that the combined capacity to block COXs and inhibit both inflammation-induced expression and activity of ASICs present in nociceptors is an important factor in the action of NSAIDs against pain.

Dependence of the acid-sensitive ion channel, ASIC1a, on extracellular Ca(2+) ions.

The early H(+)-induced current in the embryonic spinal cord neurone depends on extracellular Ca(2+) for its function. We have studied the Ca(2+)-dependence of homo- and heteromultimeric acid-sensing ion channels (ASICs) expressed in Cos cells. It was found that single-channel conductance of both the ASIC2a and the ASIC1a channel is reduced at membrane potentials more negative than -40 mV by elevated extracellular Ca(2+). Due to this effect on unitary currents, the macroscopic ASIC2a peak current at -60 mV decreases gradually with rising extracellular Ca(2+) concentration. In addition, the macroscopic ASIC1a current is very small at low extracellular Ca(2+) and increases with rising Ca(2+) up to 5 mM before decreasing again at still higher concentrations of extracellular Ca(2+).

Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels.

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H(+)-gated Na(+) channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC(50) = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

H(+)-gated cation channels.

H(+)-gated cation channels are members of a new family of ionic channels, which includes the epithelial Na+ channel and the FMRFamide-activated Na+ channel. ASIC, the first member of the H(+)-gated Na+ channel subfamily, is expressed in brain and dorsal root ganglion cells (DRGs). It is activated by pHe variations below pH 7. The presence of this channel throughout the brain suggests that the H+ might play an essential role as a neurotransmitter or neuromodulator. The ASIC channel is also present in dorsal root ganglion cells, as is its homolog DRASIC, which is specifically present in DRGs and absent in the brain. Since external acidification is a major factor in pain associated with inflammation, hematomas, cardiac or muscle ischemia, or cancer, these two channel proteins are potentially central players in pain perception. ASIC activates and inactivates rapidly, while DRASIC has both a fast and sustained component. Other members of this family such as MDEG1 and MDEG2 are either H(+)-gated Na+ channels by themselves (MDEG1) or modulators of H(+)-gated channels formed by ASIC and DRASIC. MDEG1 is of particular interest because the same mutations that produce selective neurodegeneration in C. elegans mechanosensitive neurons, when introduced in MDEG1, also produce neurodegeneration. MDEG2 is selectively expressed in DRGs, where it assembles with DRASIC to radically change its biophysical properties, making it similar to the native H(+)-gated channel, which is presently the best candidate for pain perception.

The pre-transmembrane 1 domain of acid-sensing ion channels participates in the ion pore.

The acid-sensing ion channel (ASIC) subunits ASIC1, ASIC2, and ASIC3 are members of the amiloride-sensitive Na+ channel/degenerin family of ion channels. They form proton-gated channels that are expressed in the central nervous system and in sensory neurons, where they are thought to play an important role in pain accompanying tissue acidosis. A splice variant of ASIC2, ASIC2b, is not active on its own but modifies the properties of ASIC3. In particular, whereas most members of the amiloride-sensitive Na+ channel/degenerin family are highly selective for Na+ over K+, ASIC3/ASIC2b heteromultimers show a nonselective component. Chimeras of the two splice variants allowed identification of a 9-amino acid region preceding the first transmembrane (TM) domain (pre-TM1) of ASIC2 that is involved in ion permeation and is critical for Na+ selectivity. Three amino acids in this region (Ile-19, Phe-20, and Thr-25) appear to be particularly important, because channels mutated at these residues discriminate poorly between Na+ and K+. In addition, the pH dependences of the activity of the F20S and T25K mutants are changed as compared with that of wild-type ASIC2. A corresponding ASIC3 mutant (T26K) also has modified Na+ selectivity. Our results suggest that the pre-TM1 region of ASICs participates in the ion pore.

Identification, functional expression and chromosomal localisation of a sustained human proton-gated cation channel.

Non-inactivating or slowly inactivating proton-gated cation channels are thought to play an important role in the perception of pain that accompanies tissue acidosis. We have identified a novel human proton-gated cation channel subunit that has biphasic desensitisation kinetics with both a rapidly inactivating Na+-selective and a sustained component. The protein shares 84% sequence identity with the proton-gated cation channel rASIC3 (rDRASIC) from rat sensory neurones. The biphasic desensitisation kinetics and the sequence homology suggest that this novel clone (hASIC3) is the human orthologue of rASIC3 (rDRASIC). While rASIC3 (rDRASIC) requires very acidic pH (pH < 4.5) for activation of the sustained current, the non-inactivating hASIC3 current starts to be activated when the pH decreases to below pH 6. hASIC3 is an acid sensor and might play an important role in the detection of lasting pH changes in human. We localised the hASIC3 gene to the human chromosome 7q35, 6.4 cRad telomeric from the microsatellite AFMA082XC9.

A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells.

MDEG1 is a cation channel expressed in brain that belongs to the degenerin/epithelial Na+ channel superfamily. It is activated by the same mutations which cause neurodegeneration in Caenorhabditis elegans if present in the degenerins DEG-1, MEC-4, and MEC-10. MDEG1 shares 67% sequence identity with the recently cloned proton-gated cation channel ASIC (acid sensing ion channel), a new member of the family which is present in brain and in sensory neurons. We have now identified MDEG1 as a proton-gated channel with properties different from those of ASIC. MDEG1 requires more acidic pH values for activation and has slower inactivation kinetics. In addition, we have cloned from mouse and rat brain a splice variant form of the MDEG1 channel which differs in the first 236 amino acids, including the first transmembrane region. This new membrane protein, which has been called MDEG2, is expressed in both brain and sensory neurons. MDEG2 is activated neither by mutations that bring neurodegeneration once introduced in C. elegans degenerins nor by low pH. However, it can associate both with MDEG1 and another recently cloned H+-activated channel DRASIC to form heteropolymers which display different kinetics, pH dependences, and ion selectivities. Of particular interest is the subunit combination specific for sensory neurons, MDEG2/DRASIC. In response to a drop in pH, it gives rise to a biphasic current with a sustained current which discriminates poorly between Na+ and K+, like the native H+-gated current recorded in dorsal root ganglion cells. This sustained current is thought to be required for the tonic sensation of pain caused by acids.

The potassium channel opener (-)-cromakalim prevents glutamate-induced cell death in hippocampal neurons.

(-)-Cromakalim, a typical K+-channel opener, prevents neuronal death induced by either glucose and oxygen privation or by high (100 microM) extracellular glutamate in primary cultures of hippocampus. (-)-Cromakalim has no effect on the earliest events associated with exposure to glutamate. It does not prevent the rapid rise of intracellular Ca2+, the initial swelling of neurons, or the induction of c-fos mRNA transcription. (-)-Cromakalim inhibits all delayed effects associated with the excitotoxic effect of glutamate: (a) (-)-cromakalim inhibits the late and major phase of intracellular Ca2+ increase occurring up to hours after glutamate application; and (b) although (-)-cromakalim cannot prevent the initial cellular swelling induced by glutamate, cells that have been pretreated with (-)-cromakalim return to their original size in a few hours, whereas non-(-)-cromakalim-treated cells remain swollen for more prolonged periods. Many neurons surviving the initial necrotic phase of glutamate-induced cell death undergo progressive DNA cleavage leading to apoptosis. This apoptotic process is prevented completely by (-)-cromakalim. Glibenclamide, a potent blocker of the ATP-sensitive K+ channel, abolishes all the beneficial effects of (-)-cromakalim. These findings strongly suggest that (-)-cromakalim has postsynaptic effects that are closely related to the regulation of Ca2+ homeostasis and cell volume.

The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties.

Proton-gated cation channels are acid sensors that are present in both sensory neurons and in neurons of the central nervous system. One of these acid-sensing ion channels (ASIC) has been recently cloned. This paper shows that ASIC and the mammalian degenerin MDEG, which are colocalized in the same brain regions, can directly associate with each other. Immunoprecipitation of MDEG causes coprecipitation of ASIC. Moreover, coexpression of ASIC and MDEG subunits in Xenopus oocytes generates an amiloride-sensitive H+-gated Na+ channel with novel properties (different kinetics, ionic selectivity, and pH sensitivity). In addition, coexpression of MDEG with mutants of the ASIC subunit can create constitutively active channels that become completely nonselective for Na+ versus K+ and H+-gated channels that have a drastically altered pH sensitivity compared with MDEG. These data clearly show that ASIC and MDEG can form heteromultimeric assemblies with novel properties. Heteromultimeric assembly is probably used for creating a diversity of H+-gated cation channels acting as neuronal acid sensors in different pH ranges.

Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons.

We have cloned and expressed a novel proton-gated Na+ channel subunit that is specific for sensory neurons. In COS cells, it forms a Na+ channel that responds to a drop of the extracellular pH with both a rapidly inactivating and a sustained Na+ current. This biphasic kinetic closely resembles that of the H+-gated current described in sensory neurons of dorsal root ganglia (1). Both the abundance of this novel H+-gated Na+ channel subunit in sensory neurons and the kinetics of the channel suggest that it is part of the channel complex responsible for the sustained H+-activated cation current in sensory neurons that is thought to be important for the prolonged perception of pain that accompanies tissue acidosis (1, 2).

Comparative expression of the inward rectifier K+ channel GIRK2 in the cerebellum of normal and weaver mutant mice.

The main target for degeneration associated with the weaver mutation is the cerebellum. Expression of the GIRK2 mRNA and protein was studied in cerebellum of 12- and 22-day-old normal and weaver mice. In 12-day-old mice, GIRK2 is expressed at highest levels in the external granule layer (EGL) and in lower levels in the newly forming internal granule layer (IGL). In the weaver cerebellum, a high hybridization signal and dark immunostaining was observed in the EGL due to the higher density of non-migrated cells. In 22-day-old weaver cerebella, there are only few remaining granule cells existing as scattered cells within the IGL and molecular layer. GIRK2 is expressed in these neurons but the majority of cells expressing GIRK2 in these cerebella are Purkinje cells that are also affected by the weaver mutation (position, shape) but have not died. Normal cerebellar granule neurons but not homozygous mutant neurons in primary cultures and cerebellar slices of 8-day-old mice displayed inward rectifier K+ currents. Taken together, these findings suggest that cell loss in the weaver cerebellum is not directly related to a differential content of GIRK2 in the affected neurons during development. The lethal effect of the weaver mutation in specific neurons is probably due to a combination of the abnormal function of the inward rectifier K+ channels and other factors specific to the vulnerable neurons.

Modes of regulation of shab K+ channel activity by the Kv8.1 subunit.

The Kv8.1 subunit is unable to generate K+ channel activity in Xenopus oocytes or in COSm6 cells. The Kv8.1 subunit expressed at high levels acts as a specific suppressor of the activity of Kv2 and Kv3 channels in Xenopus oocytes (Hugnot, J. P., Salinas, M., Lesage, F., Guillemare, E., Weille, J., Heurteaux, C., Mattéi, M. G., and Lazdunski, M. (1996) EMBO J. 15, 3322-3331). At lower levels, Kv8.1 associates with Kv2.1 and Kv2.2 to form hybrid Kv8.1/Kv2 channels, which have new biophysical properties and more particularly modified properties of the inactivation process as compared with homopolymers of Kv2.1 or Kv2.2 channels. The same effects have been seen by coexpressing the Kv8.1 subunit and the Kv2.2 subunit in COSm6 cells. In these cells, Kv8.1 expressed alone remains in intracellular compartments, but it can reach the plasma membrane when it associates with Kv2.2, and it then also forms new types of Kv8.1/Kv2. 2 channels. Present results indicate that Kv8.1 when expressed at low concentrations acts as a modifier of Kv2.1 and Kv2.2 activity, while when expressed at high concentrations in oocytes it completely abolishes Kv2.1, Kv2.2, or Kv3.4 K+ channel activity. The S6 segment of Kv8.1 is atypical and contains the structural elements that modify inactivation of Kv2 channels.

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1-CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3-8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N-terminal domain.

Functional receptors in Xenopus oocytes for U-37883A, a novel ATP-sensitive K+ channel blocker: comparison with rat insulinoma cells.

Follicle-enclosed Xenopus oocytes were used to describe the ATP-sensitive K+ (KATP) channel-blocking properties of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl), in comparison with glibenclamide. In follicular oocytes, the KATP channel opener P1060 (30 microM), a pinacidil analog, activated a large outward K+ current that was blocked by glibenclamide (IC50 = 0.33 microM) and U-37883A (IC50 = 0.26 microM). P1060 activation was inhibited by both U-37883A and glibenclamide in a noncompetitive manner. U-37883A also blocked the KATP channel activation by cAMP (300 microM) and adenosine (10 microM). Single-channel studies on isolated follicular cells showed that U-37883A (10 microM) reduced the open probability of the KATP channel by 76%, without significantly modifying the single-channel current amplitude. Receptor binding studies with [3H]U-37883 in membranes from follicle-enclosed oocytes demonstrated a single class of low affinity binding sites, with a Kd of 450 nM and a Bmax of 17 pmol/mg of protein. Studies with analogs of U-37883A showed that U-52090A inhibited KATP current and displaced [3H]U-37883 from its binding site with similar potencies. In contrast, U-42069D neither inhibited KATP current nor competed with [3H]U-37883 binding. In RINm5F cells (an insulinoma cell line), U-37883A, unlike glibenclamide, failed to inhibit KATP current. Furthermore, there was no significant specific binding of [3H]U-37883 in RINm5F cell membranes, which displayed high levels of specific binding of [3H]glibenclamide. These data demonstrate the presence of a receptor for U-37883A-type guanidines that controls the activity of the endogenous KATP channels in follicle-enclosed oocytes. The available data collectively suggest that U-37883A is a more selective blocker of the follicular KATP channel, which is very similar to that in smooth muscle, than of the pancreatic beta cell KATP channel.

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.

Effectors of ATP-sensitive K+ channels inhibit the regulatory effects of somatostatin and GH-releasing factor on growth hormone secretion.

Somatostatin inhibition of growth hormone (GH) secretion from adenohypophysis cells in culture was antagonized by the antidiabetic sulfonylurea glipizide (K0.5 = 10 +/- 5 nM). Although all cells that hyperpolarize with somatostatin have ATP-sensitive K+ channels, the antagonistic actions of the hormone and of the antidiabetic drug are due to effects on different types of K+ channels. Diazoxide, an opener of ATP-sensitive K+ channels, abolished the increase of intracellular Ca2+ provoked by growth hormone releasing factor (GRF) and induced inhibition of GRF stimulated GH secretion (K0.5 = 138 microM). This inhibition by diazoxide was largely suppressed by glipizide which blocked the ATP-sensitive K+ channels opened by diazoxide. In summary, hormonal activation of GH secretion is inhibited by openers of ATP-sensitive K+ channels, while hormonal inhibition of GH secretion is suppressed by blockers of ATP-sensitive K+ channels.