Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Δg _m , was initially determined. DT-AB induced a ∼10-fold lower Δg _m than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg _m , whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg _m . Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Δg _m and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel. Received: 20 June 1996/Revised: 8 November 1996     

Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae

The anaerobic performance of gpd1Δ and gpd2Δ mutants of Saccharomyces cerevisiae was characterized and compared to that of a wild-type strain under well-controlled conditions by using a high-performance bioreactor. There was a 40% reduction in glycerol level in the gpd2Δ mutant compared to the wild-type. Also the gpd1Δ mutant showed a slight decrease in glycerol formation but to a much lesser degree. As a consequence, ethanol formation in the gpd2Δ mutant was elevated by 13%. In terms of growth, the gpd1Δ mutant and the wild-type were indistinguishable. The gpd2Δ mutant, on the other hand, displayed an extended lag phase as well as a reduced growth rate under the exponential phase. Even though glycerol-3-phosphate dehydrogenase 2 (GPD2) is the important enzyme under anaerobic conditions it can, at least in part, be substituted by GPD1. This was indicated by the higher expression level of GPD1 in the gpd2Δ mutant compared to the wild type. These results also show that the cells are able to cope and maintain redox balance under anaerobic conditions even if glycerol formation is substantially reduced, as observed in the gpd2Δ mutant. One obvious way of solving the redox problem would be to make a biomass containing less protein, since most of the excess NADH originates from amino acid biosynthesis. However, the gpd2Δ mutant did not show any decrease in the protein content of the biomass. Received: 16 February 1998 / Received revision: 16 March 1998 / Accepted: 1 June 1998     

Roles of <Emphasis Type="Italic">Saccharomyces cerevisiae RAD17</Emphasis> and <Emphasis Type="Italic">CHK1</Emphasis> checkpoint genes in the repair of double-strand breaks in cycling cells

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Δ/chk1Δ and rad17Δ/rad17Δ, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Δ/rad17Δ cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Δ/rad17Δ cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Δ/chk1Δ cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Δ/chk1Δ mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background. Nelson Bracesco and Ema C. Candreva have contributed equally to this article.     

Inward and outward rectifying potassium currents inSaccharomyces cerevisiae mediated by endogenous and heterelogously expressed ion channels

Disruption of genes encoding endogenous transport proteins inSaccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K⁺ channels and amino acid transporters from the plantArabidopsis thaliana [1–4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability oftrk1Δtrk2Δ mutants ofS. cerevisiae to grow on submillimolar K⁺ correlates with the lack of K⁺ inward currents, which are present in wild-type cells, and that transformation of thetrk1Δtrk2Δ double-deletion mutant withKAT1 fromArabidopsis thaliana restores this phenotype by encoding a plasma membrane protein that allows large K⁺ inward currents. Similar K⁺ inward currents are induced by transformation of atrk1 mutant withAKT1 fromA. thaliana. This work was supported by a grant from theForschungsgemeinschaft (A.B.), TheU.S. Department of Energy (c.L.S.), The U.S. National Science Foundation (R.F.G.) Lisboa, Portugal.     

Influence of Cys-130 S. aureus Alpha-toxin on Planar Lipid Bilayer and Erythrocyte Membranes

Replacement of an amino acid residue at position 130 -Gly by Cys- in the primary structure of Staphylococcus aureus alpha-toxin decreases the single-channel conductance induced by the toxin in planar lipid bilayers. Concomitantly, the pH value at which the channel becomes unable to discriminate between Cl⁻ and K⁺ ions is also decreased. By contrast, the pH dependence of the efficiency of the mutant toxin to form ion channels in lipid bilayers was unchanged (maximum efficiency at pH 5.5–6.0). The asymmetry and nonlinearity of the current-voltage characteristics of the channel were increased by the point mutation but the diameter of the water pore induced by the mutant toxin, evaluated in lipid bilayers and in erythrocyte membranes, was found to be indistinguishable from that formed by wild-type toxin and equal to 2.4–2.6 nm. Alterations at the ``trans mouth' were found to be responsible for all observed changes of the channel properties. This mouth is situated close to the surface of the second leaflet of a bilayer lipid membrane. The data obtained allows us to propose that the region around residue 130 in fact determines the main features of the ST-channel and takes part in the formation of the trans entrance of the channel. Received: 8 September 1995/Revised: 20 November 1996     

Transport of large organic ions through syringomycin channels in membranes containing dipole modifiers

The effect of the membrane dipole potential (φ _d ) on conductance and the steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate, and chloride was shown. The φ _d value varied with the introduction of phloretin to membrane bathing solutions, which reduces φ _d and RH 421, which increases φ _d . It was established that, in all studied systems, an increase in the membrane dipole potential caused a decrease in the steady-state number of open channels. In systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are one order lower than those of systems that contain sodium chloride. At the same time, the conductance (g) of single SRE channels in the membranes bathed in NaCl solution increases with increase in φ _d and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the φ _d . The latter is due to the lack of cation/anion selectivity of the SRE channels in these systems. The different channel-forming activity of SRE in the experimental systems is determined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

Rapid kinetics of glucose uptake inSaccharomyces cerevisiae

In a multiple deletion mutanthxt1Δhxt2Δhxt3Δ hxt4Δsnf3Δ ofSaccharomyces cerevisiae growing on 2 % glucose, high-affinity glucose-uptake (lowK _m) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. Howver, in a mutant containing only theHXT2 gene (hxt1Δhxt3Δhxt4Δ snf3Δ), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such asHXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (K _m approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.     

Conductance of phytotoxin channels in the presence of large organic ions

In this study we evaluated the effect of the size of penetrating anions on properties of the channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes. Conductance and the mean lifetime of SRE channels were measured in 0.4 M solutions of sodium chloride, aspartate, and gluconate. A comparison of results of conductometric and electrophysiological measurements has shown the following: (1) the ratio of mobilities of aspartate anions in the channel and in the aqueous solution is five times lower than that of chlorine anions and (2) the conductance of channels in the presence of sodium gluconate is due mainly to cations. The obtained results indicate the binding of penetrating anions to the selective filter of the SRE pore. The radius of the SRE channel selective filter has been established (r ∼ 0.3 nm) and its localization in the cis-mouth of the pore has been found.     

Giant yeast cells with nonrecyclable ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thereby provides the precursors required for DNA synthesis and repair. In an attempt to test cell resistance to a permanent replicational stress, we constructed a mutant Saccharomyces cerevisiae strain containing exclusively nonrecyclable catalytic subunits of RNR that become inactivated following the reduction of one ribonucleoside diphosphate. In this rnr1C883A rnr3Δ mutant, the synthesis of each deoxyribonucleotide thus requires the production of one Rnr1C883A protein, which means that 26 million Rnr1C883A proteins (half the protein complement of a wild-type cell) have to be produced during each cell cycle. rnr1C883A rnr3Δ cells grow under constant replicational stress, as evidenced by the constitutive activation of the checkpoint effector Rad53, and their S phase is considerably extended compared to the wild type. rnr1C883A rnr3Δ mutants also display additional abnormalities such as a median cell volume increased by a factor of 8, and the presence of massive inclusion bodies. However, they exhibit a good plating efficiency and can be propagated indefinitely. rnr1C883A rnr3Δ cells, which can be used as a protein overexpression system, thus illustrate the robustness of S. cerevisiae to multiple physiological parameters.     

Deletion of BGL2 results in an increased chitin level in the cell wall of Saccharomyces cerevisiae

It is shown that the deletion of BGL2 gene leads to increase in chitin content in the cell wall of Saccharomyces cerevisiae. A part of the additional chitin can be removed from the bgl2Δ cell wall by alkali or trypsin treatment. Chitin synthase 1 (Chs1) activity was increased by 60 % in bgl2Δ mutant. No increase in chitin synthase 3 (Chs3) activity in bgl2Δ cells was observed, while they became more sensitive to Nikkomycin Z. The chitin level in the cell walls of a strain lacking both BGL2 and CHS3 genes was higher than that in chs3Δ and lower than that in bgl2Δ strains. Together these data indicate that the deletion of BGL2 results in the accumulation and abnormal incorporation of chitin into the cell wall of S. cerevisiae, and both Chs1 and Chs3 take part in a response to BGL2 deletion in S. cerevisiae cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic

The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2–2.5-fold upon addition of either arsenate (As^V) or arsenite (As^III). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3′-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation.     

High-cell-density cultivation for co-production of ergosterol and reduced glutathione by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Two different high-cell-density cultivation processes based on the mutant Saccharomyces cerevisiae GE-2 for simultaneous production of glutathione and ergosterol were investigated. Compared with keeping the ethanol volumetric concentration at a constant low level, feedback control of glucose feeding rate (F) by keeping the descending rate of ethanol volumetric concentration (ΔE/Δt) between −0.1% and 0.15% per hour was much more efficient to achieve a high glutathione and ergosterol productivity. This bioprocess overcomes some disadvantages of traditional S. cerevisiae-based cultivation process, especially shortening cultivation period and making the cultivation process steady-going. A classical on or off controller was used to manipulate F to maintain ΔE/Δt at its set point. The dry cell weight, glutathione yield and ergosterol yield reached 110.0 ± 2.6 g/l, 2,280 ± 76 mg/l, and 1,510 ± 28 mg/l in 32 h, respectively.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k _cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.     

转录因子PaPho与丝状真菌Podospora anserina中磷元素的吸收和利用研究

作为生物体必需的营养元素之一,磷在物质代谢、信号传导和能量储存中起着关键作用。【目的】研究丝状真菌Podospora anserina中调控磷酸盐代谢相关转录因子的作用,可进一步阐明真核微生物中磷元素吸收的调控机制。【方法】利用同源重组的方法定点敲除P.anserina中2个磷代谢相关转录因子PaPho1和PaPho2,遗传杂交构建双重突变体ΔPaPho1ΔPaPho2;通过表型分析、无机磷含量测定和酸性磷酸酶活性测定分析各突变菌株的变化;利用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)分析磷代谢相关基因的表达情况。【结果】在无机磷作为唯一磷来源的培养基上,ΔPaPho1ΔPaPho2无法生长;在添加有机磷的培养基中,ΔPaPho1ΔPaPho2和野生型菌株生长无显著性差异。在同时添加有机磷和无机磷的培养基中,ΔPaPho1ΔPaPho2的无机磷含量和酸性磷酸酶活性比野生型菌株的分别下降了25.0%和61.9%,ΔPaPho1ΔPaPho2中无机磷酸盐转运蛋白基因的表达水平显著降低。【结论】在P...     

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL–lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL–lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of β-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The ΔmscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase ΔmscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, ΔmscL cells had become resistant to a 0.9 M osmotic downshift.     

The RAD3 gene of Saccharomyces cerevisiae: isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant

Summary Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2–4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or radiation.     

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Reconstitution in Lipid Bilayers of an ATP-Sensitive K+ Channel from Pig Coronary Smooth Muscle

A K⁺ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K⁺ channel (K_ATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na₂. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the K_ATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, K_ATP channels were characterized by brief openings followed by long closings (open probability, P _o ≈ 0.02); only occasionally (3 out of 12 experiments) did the K_ATP channels have a high open probability (P _o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl₂ to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996