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.

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.

8-Methoxypsoralen blocks ATP-sensitive potassium channels and stimulates insulin release.

8-Methoxypsoralen (8-MOP) stimulated insulin release from HIT-T15 B-cells and inhibited the diazoxide-induced and sulfonylurea-sensitive 86Rb+ efflux from these cells. These results indicate that 8-MOP affects ATP-sensitive K+ channel activity. Patch-clamp experiments confirmed this view.

TMB-8 (8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate) inhibits the ATP-sensitive K+ channel.

Whole-cell current clamp, single-channel recordings and 86Rb+ flux techniques have been used to show that 8-(N,N-diethyl-amino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibits ATP-sensitive K+ channels in HIT-T15 beta-cells. TMB-8 inhibition is observed when KATP channels are activated by ATP depletion or by the K+ channel opener, diazoxide.

ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids.

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.

Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.

Chlorpromazine and related phenothiazines inhibit the ATP-sensitive K+ channel.

Whole-cell, current clamp, single channel recordings and 86Rb+ flux techniques were used to show that phenothiazines inhibit ATP-sensitive K+ channels (KATP) in HIT-T15 beta-cells. Chlorpromazine inhibition was observed when KATP channels were activated by ATP depletion or by direct treatment with a classical KATP channel opener, diazoxide. The order of potency of the phenothiazines tested was chlorpromazine greater than triflupromazine greater than fluphenazine greater than trifluopromazine with IC50 values of 1, 4, 6 and 20 microM, respectively. The inhibition was reversible.

Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.

The venom of the black mamba contains a 60-amino acid peptide called calciseptine. The peptide has been fully sequenced. It is a smooth muscle relaxant and an inhibitor of cardiac contractions. Its physiological action resembles that of drugs, such as the 1,4-dihydropyridines, which are important in the treatment of cardiovascular diseases. Calciseptine, like the 1,4-dihydropyridines, selectively blocks L-type Ca2+ channels and is totally inactive on other voltage-dependent Ca2+ channels such as N-type and T-type channels. To our knowledge, it is the only natural polypeptide that has been shown to be a specific inhibitor of L-type Ca2+ channels.

Activation by cromakalim of pre- and post-synaptic ATP-sensitive K+ channels in substantia nigra.

Membrane potentials and synaptic potentials were recorded using the patch clamp technique from neurons isolated from the substantia nigra. Intracellular perfusion of dopaminergic neurons with an ATP-free solution caused hyperpolarization and inhibition of firing. Intracellular perfusion with a solution containing 2 mM ATP prevented this hyperpolarization, but application of the K+ channel openers cromakalim and pinacidil caused a similar hyperpolarization as well as the disappearance of bicuculline-sensitive synaptic potentials. All these effects were reversed by sulfonylureas, indicating that they are mediated by ATP-sensitive K+ channels. It is concluded that K+ channel openers activate ATP-sensitive K+ channels both presynaptically on GABAergic terminals and postsynaptically on substantia nigra dopaminergic neurons.

ATP-sensitive K+ channels reveal the effects of intracellular chloride variations on cytoplasmic ATP concentrations and mitochondrial function.

Replacement of intracellular Cl- by impermeant anions, as well as treatment of insulinoma cells by the Cl- channel blocker, NPPB, leads to activation of ATP-dependent K+ (KATP) channels. Activation of KATP channels by C1- substitution is eliminated (i) when intracellular ATP is replaced by non-hydrolyzable ATP analogs, (ii) when the perfusion medium contains an ATP regenerating system, (iii) when the mitochondrial ATPase is blocked by oligomycin. Dinitrophenol and GDP have the same activating effects on KATP channels as NPPB or intracellular Cl- substitution. Our interpretation of the results is that NPPB and intracellular Cl- replacement produce an uncoupling of oxidative phosphorylation by acting on mitochondrial anion channels, which leads to rapid degradation of ATP and to activation of KATP channels. KATP channels are useful sensors of cytoplasmic ATP variations.