Intracellular acidification does not account for inhibition of Saccharomyces cerevisiae growth in the presence of ethanol

Abstract Intracellular acidification has been considered one of a number of mechanisms underlying the inhibition of growth and fermentation by ethanol in yeast. However, most of the studies on the effect of ethanol on yeast intracellular pH (pH_i) were carried out by using unadapted cells to which ethanol was added. In this paper we show that the pH_i of exponential cells of Saccharomyces cerevisiae IGC 3507 III grown in a medium with glucose and inhibitory concentrations of ethanol only decreased to values below those in unstressed cells (6.9) for concentrations equal to or above 7% (v/v). Only at these supracritical levels (7–10% (v/v)) was pH homeostasis in ethanol-adapted yeast affected. This is consistent with the significant increase of plasma membrane permeability and decrease of plasma membrane H⁺-ATPase in comparison with the corresponding values in unstressed cells. These deleterious effects were only observed with those high concentrations of toxin. These results indicate that intracellular acidification does not account for inhibition of yeast growth in the presence of ethanol. In fact, growth was inhibited by ethanol concentrations (3–6% (v/v)) that did not lead to the decrease of pH_i. Furthermore, even for supracritical concentrations, close to the maximal that allowed growth (10% (v/v)), the dedrease of pH_i was not important reaching, at the most, values of 6.5–6.6.     

The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method

The change in intracellular pH (pH_i) upon fertilization and the effects of changing the pH_i by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pH_i was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pH_i ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pH_i ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1–4), development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pH_i did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pH_i was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pH_i. The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.     

Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca²⁺ and intracellular pH (pH_i). Since the two jelly components essential for inducing the acrosome reaction, ARIS and Co-ARIS, were shown to activate Ca-channels (accompanying paper), we examined the jelly components to determine which was responsible for the pH_i-increase using 9-aminoacridine as a probe of pH_i. This paper presents evidence that an oligopeptide(s) is responsible for the pH_i-increase. The pH_i of swimming sperm is 7.4-7.5. Within 20 sec after the addition of jelly, their pH_i increased rapidly by 0.06 pH unit, then decreased by 0.2–0.3 pH unit, and reached a plateau level within 3 min. Similar changes in pH_i were observed on addition of a Pronase digest of ARIS (P-ARIS) and a diffusible fraction of jelly (Fraction M₈) together. Fraction M₈, but not ARIS or Co-ARIS increased the pH_i, and activated sperm respiration in sea water at pH 6.5. The two activities of Fraction M₈ depended upon Na⁺ but not Ca²⁺, and were susceptible to Pronase digestion. Fraction M₈ is also known to enhance induction of the acrosome reaction by the Ca-ionophore A23187. These results suggest that the egg jelly contains a peptide(s) that is not obligatory for the acrosome reaction but facilitates the reaction by increasing the pH_i of the sperm. The significance of the pH_i-increase upon the acrosome reaction is discussed.     

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus . FSG and/or SAP-I induced elevations of [Ca²⁺]; and pH_i in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca²⁺]_i or pH_i of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca²⁺]_i and pH_i, At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca²⁺]_i, although SAP-I elevated the pH_i, its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K⁺-channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca²⁺]_i and pH_i by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca²⁺ and H⁺ transport systems.     

Effects of Extracellular Potassium on Acid Release and Motility Initiation in Toxoplasma gondii

The internal pH (pH_i) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pH_i of the metabolizing parasite increased when the extracellular K⁺ was elevated in alkaline medium or when the external pH (pH_c) was substantially increased in medium employing high external K⁺ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pH_i was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pH_c was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.     

Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein

In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na⁺/H⁺ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na⁺/H⁺ exchanger nor a proliferation-associated increase in intracellular pH (pH_i) has been demonstrated.
The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na⁺/H⁺ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pH_i measurements in BALB/MK cells. Initial measurements of the pH_i using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes.
Using SNARF-calcein we were able to show proliferation to be associated with increased pH_i. However, culture conditions were critical for these measurements. Our data indicate that the Na⁺/H⁺ exchanger is involved in this process, since acid load and pH_i-recovery experiments showed the alkalization to be amiloride-sensitive.     

Adrenergic responses and the Root effect in erythrocytes of freshwater fish

The effects of adrenergic-stimulation upon the oxygen-binding capacity of fish erythrocytes have been investigated. The oxygen capacity of rainbow trout, Oncorhynchus mykiss (Walbaum), erythrocytes was lowered by 44% on extracellular acidification (the so-called 'Root effect'). Addition of isoproterenol at 20° Ccaused an acid shift of the curve relating oxygen capacity to pH₀ by approximately 0.2 pH units, a value which was similar to the change in intracellular pH caused by adrenergic stimulation (Cossins & Kilbey Journal of Experimental Biology , 148 , 303–312, 1990). Moreover, when plotted as a function of pH_i, the curves for control and adrenalinstimulated erythrocytes were superimposable suggesting that the adrenergic shift in the Root curve was a result of the change in pH_i caused by activation of the adrenergic Na⁺/H⁺ exchanger.
A similarly large adrenergic shift in the Root curve was observed for pike, Esox lucius L., erythrocytes, though not for erythrocytes of carp, Cyprinus carpio L., and tench, Tinea tinea (L.). The pH for the mid-point of the Root effect in pike erythrocytes was distinctly more acid than for trout, but in both cases corresponded closely with the optimal pH for the adrenergic Na⁺/H⁺ exchange mechanism. This suggests a link between the functional characteristics of the exchanger and the oxygen-binding properties of haemoglobin.     

3-Hydroxybutyrate Aids the Recovery of the Energy State from Aglycaemic Hypoxia of Adult but Not Neonatal Rat Brain Slices

Abstract: The level of phosphocreatine (PCr) and the intracellular pH (pH_i) of superfused cortical brain slices from adult or 10-day-old rats were monitored using ³¹P NMR. When the glucose in the superfusing medium was replaced by 3-hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pH_i was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pH_i returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.     

Effects of pH on the Fertilization Response of the Medaka Egg

The effects of changing the intra- and extracellular pH on the cortical reaction and sperm penetration into eggs were examined in Oryzias latipes. When eggs were inseminated in a saline adjusted to various pH values with different buffers, fertilization took place normally over a wide range of external pH (pH₀) from about 6 to 10 with a peak at 7 to 8.5. The primary causes of the reduced fertilization were impaired motility or immobility of the spermatozoa in the acidic saline and the swelling of the chorion in the alkaline saline. A rise in the intracellular pH (pH_i) shortly after commencement of the cortical reaction was found by monitoring the color of a pH indicator micro-injected into the cytoplasm. The experimental results obtained by microinjection of various pH buffers (pH 4–12) suggest that the intracellular membrane fusion of the plasma membrane with the cortical alveolar membrane (CABD) is affected by the acidic pH_i while the propagation of the CABD and the intercellular membrane fusion between the plasma membranes of the egg and the spermatozoon are affected by the acidic pH₀.     

Acid sensitivity induction (ASI) at alkaline pH in Escherichia coli involves two major sensitization components, induction of both being switched on by increased internal pH

Acid sensitivity induction (ASI) at alkaline pH in Escherichia coli 1157 ( phoE ) is only fully mounted if protein synthesis occurs for the first 5 min of the 12–15 min induction period, but appreciable sensitization occurs in the presence of chloramphenicol indicating that there are protein synthesis-independent and -dependent components (components 1 and 2). Component 1 sensitization is in place after 10 min at pH_o 9.0 but the dependent process is induced slightly more slowly. Collapsing ΔpH at pH_o 9.0 did not prevent full ASI, indicating that component 1 and 2 induction does not depend on increased ΔpH. The two induction (or activation) processes responded differently to intermediate levels of external alkalinization; component 1 was induced at pH_o 8.3 but component 2 needed pH_o 8.4 or greater. Collapsing ΔpH at pH_o 8.0 led to full induction of component 1 and appreciable induction of component 2, indicating that both processes are triggered if pH_i rises to 8.0 or greater (presumably at pH_o 8.3–8.4). Component 1 appears less important for ASI in 1157-4 ( phoE ⁺) than in 1157 ( phoE ).     

Parthenogenesis in Urechis caupo (Echiura) II. Role of intracellular pH in parthenogenesis induction

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo . We have shown previously that eggs exposed to P23 for ≥3min complete meiosis but fail to cleave. However, a brief (1.5–2 min) exposure to P23 at pH 8, followed by either acidification of the seawater to pH 7 or dilution of P23 at pH 8 causes germinal vesicle breakdown (GVBD), but eggs fail to complete meiosis and many then later advance to mitosis. In the present study we investigated the hypothesis that partial activation leading to parthenogenesis occurs when there is a partial intracellular alkalinization. Measurements with the fluorescent pH indicator bis(carboxyethyl)-carboxyfluorescein (BCECF) showed that P23 induces a pH, increase similar to that occurring during fertilization and that parthenogenesis-inducing treatments interupt this rise in pH₁ In eggs exposed to P23 for >3 min the pH₁ increase was 0.31–0.49 units, slightly higher than in fertilized eggs. In partially activated eggs exposed to P23 for 1.5–2 min at pH 8, pH₁ began to rise but then returned to control values or remained only partially elevated (< 0.2 pH units average increase). Electrophysiological measurements revealed that removal of P23 during the first few minutes of exposure caused the activation potential to terminate and experiments with [¹⁴C]-P23 confirmed that dilution results in a rapid unbinding of P23 from eggs. If proton export is driven by membrane potential as well as the pH gradient, these results explain why dilution of P23 at pH 8 also interrupts the pH_i increase.     

Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABAB-activated GIRK channels in Purkinje cells

Activation of G protein-gated inwardly-rectifying K⁺ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA_B) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA_B receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA_B receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA_B receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA_B receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA_B receptors. The association of GIRK channels and GABA_B receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.     

The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli

R. J. ROWBURY, M. GOODSON AND A.D. WALLACE. 1992. Escherichia coli K12 becomes resistant to killing by acid (habituates to acid) in a few minutes at pH 5.0. Habituation involves protein synthesis-dependent and -independent stages; both must occur at an habituating pH. The habituation sensor does not detect increased ΔpH (or decreased Δψ) nor an increased difference between pH_o and periplasmic pH but probably detects a fall in either external or periplasmic pH. Phosphate ions inhibit habituation, at any stage, probably by interfering with outer membrane passage of hydrogen ions. Most outer membrane components tested are not required for habituation but phoE deletion mutants habituated poorly and are acid-resistant. Strains derepressed for phoE , in contrast, showed increased acid sensitivity. These and other results suggest that habituation involves hydrogen ions or protonated carriers crossing the outer membrane preferentially via the PhoE pore, a process inhibited by phosphate and other anions. Stimulation by phosphate of the poor growth of E. coli at pH 5.0 is in accord with the above. Acetate did not enhance acid killing of pH 5.0 cells, suggesting that their resistance does not depend on maintaining pH_i near to neutrality at an acidic pH_o level.     

Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.     

Effect of oxygen concentration on intracellular pH, glucose-6-phosphate and NTP content in rice (Oryza sativa) and wheat (Triticum aestivum) root tips: in vivo 31P-NMR study

Hypoxic pretreatment is known to induce anoxia tolerance in plant species sensitive to oxygen deprivation. However, we still do not have detailed information on changes in cytoplasmic and vacuolar pH (pH_cyt and pH_vac) in plants under low-oxygen availability (hypoxia) and under anoxia. To investigate this, we have studied the influence of hypoxia and anoxia on pH_cyt and pH_vac, glucose-6-phosphate (Glc-6-P) and nucleotide triphosphate (NTP) contents in rice ( Oryza sativa L.) root tips in comparison with those of wheat ( Triticum aestivum L.) with in vivo ³¹P-nuclear magnetic resonance. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units after 6 h). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt= 10^−pHcyt) for both hypoxic and anoxic conditions. Glc-6-P content increased in rice under anoxia and hypoxia. In wheat, Glc-6-P was not detectable under anoxia but increased under hypoxia. In this study, rice root tips were shown to behave as anoxia tolerant tissues. Our results suggest that the initial cytoplasmic acidification and subsequent pH_cyt are differently regulated in anoxia tolerant and intolerant plants and depend on the external oxygen concentration.     

GABABR1a/R1b-Type Receptor Antisense Deoxynucleotide Treatment of Melanotropes Blocks Chronic GABAB Receptor Inhibition of High Voltage-Activated Ca2+ Channels

Abstract: GABA_B and dopamine D₂ receptors, both of which acutely inhibit adenylyl cyclase and high voltage-activated Ca²⁺ channels (HVA-CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D₂ receptor agonist application in vitro has been reported to result in inhibition of HVA-CC activity by down-regulation. Here we report that chronic GABA_B, but not GABA_A, agonist treatment also resulted in HVA-CC inhibition. Two GABA_B receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK-293 cells. We have constructed an antisense deoxynucleotide knockdown-type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABA_B antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA-CC inhibition by the D₂ agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D₂ antisense knockdown probe eliminated the ability of a D₂ agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABA_B receptor, which, like the D₂ receptor, is coupled to chronic HVA-CC inhibition in melanotropes.     

The mechanism of bicarbonate assimilation by the polar leaves of Potamogeton and Elodea. CO2 concentrations at the leaf surface

Abstract. Photosynthetic utilization of HCO, in leaves of Poiamogeton and Elodea occurs at the lower leaf side, with subsequent OH∼ release at the upper side. It is accompanied by transport of cations, in the present experiment K +, across the leaf. The resulting pH and K+ concentration changes near the leaf surface were recorded with miniature electrodes. From the pH and K+ concentration the concentrations of the different inorganic carbon species were calculated and compared with photosynthetic O, production. HCO⁻₃ utilization is accompanied by a drastic increase in the free CO₂ concentration near the lower epidermis. Experiments with CO₂− and HCO₃⁻free solutions showed an oscillating acidification near the lower epidermis and alkalinization near the upper epidermis. It is concluded that the acidification results from the activity of light-dependent H+ pumps. The finding that an increase in pH at the upper side always coincided with a decrease at the lower in these experiments shows that the H+ pumps and the OH− extruding mechanism are coupled although occurring in different cell layers. Previously we have suggested that the first step in the process of photosynthetic HCO₃⁻ utilization is external conversion of HCO₃⁻" by acidification caused by light-dependent H+ pumps. The present results strongly support this hypothesis. Two possible pathways for the accompanying K + transport are discussed. The model presented here explains the known inhibiting effects of buffers and high pH on photosynlhetic HCO₃⁻ utilization.     

Dual Signalling by the Adenosine A2a Receptor Involves Activation of Both N- and P-Type Calcium Channels by Different G Proteins and Protein Kinases in the Same Striatal Nerve Terminals

Abstract: Many G_s-linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A_2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of G_s, adenylyl cyclase, protein kinase A, and P-type calcium channels; the other is mediated by a cholera toxin-insensitive G protein, protein kinase C, and N-type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A_2a receptor antagonist KF17837. This demonstrates that the A_2a receptor activates two signalling systems in striatal cholinergic neurons.     

Immunological Characterization and Transmembrane Topology of 5-Hydroxytryptamine3 Receptors by Functional Epitope Tagging

Abstract: 5-Hydroxytryptamine₃ (5-HT₃) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABA_A, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5-HT₃ receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5-HT₃ receptors in transfected COS-7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti-fusion protein antibodies directed against a wild-type receptor intracellular domain. The extracellular location of the distal C-terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5-HT₃ serotonin-gated channels. In this region, the significant homology between members of this class of neurotransmitter-gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate-gated and ATP-gated channels.     

The High-Affinity Sulphonylurea Receptor Regulates KATP Channels in Nerve Terminals of the Rat Motor Cortex

Abstract: The coexpression of sulphonylurea binding sites and ATP-sensitive K⁺ (K_ATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea-sensitive K_ATP channels and charybdotoxin-sensitive, large-conductance calcium-activated K⁺ BK_Ca channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high-affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium-independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and K_ATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the K_ATP channel may serve to limit glutamate release under conditions of metabolic stress.