Genetically engineered metal ion binding sites on the outside of a Channel's transmembrane beta-barrel

We are exploring the ability of genetically engineered versions of the Staphylococcus aureus alpha-hemolysin (alphaHL) ion channel to serve as rationally designed sensor components for analytes including divalent cations. We show here that neither the hemolytic activity nor the single channel current of wild-type alphaHL was affected by [Zn(II)] 相似文献   

Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons: role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 subunits

N-Methyl-D-aspartate (NMDA) receptors are modulated by extracellular spermine and protons and are blocked in a voltage-dependent manner by spermine and polyamine derivatives such as N1-dansyl-spermine (N1-DnsSpm). The effects of mutations in the first and third transmembrane domains (M1 and M3) and the pore-forming loop (M2) of NMDA receptor subunits were studied. Surprisingly, some mutations in M2 and M3 of the NR1 subunit, including mutations at W608 and N616 in M2, reduced spermine stimulation and proton inhibition. These mutations may have long-range allosteric effects or may change spermine- and pH-dependent gating processes rather than directly affecting the binding sites for these modulators because spermine stimulation and proton inhibition are not voltage dependent and are thought to involve binding sites outside the pore-forming regions of the receptor. A number of mutations in M1-M3, including mutations at tryptophan and tyrosine residues near the extracellular sides of M1 and M3, reduced block by spermine and N1-DnsSpm. The effects of these mutants on channel block were characterized in detail by using N1-DnsSpm, which produces block but not stimulation of NMDA receptors. Block by N1-DnsSpm was studied by using voltage ramps analyzed with the Woodhull model of channel block. Mutations at W563 (in M1) and E621 (immediately after M2) in the NR1A subunit and at Y646 (in M3) and N616 (in the M2 loop) in the NR2B subunit reduced the affinity for N1-DnsSpm without affecting the voltage dependence of block. These residues may form part of a binding site for N1-DnsSpm. Mutation of a tryptophan residue at position W607 in the M2 region of NR2B greatly reduced block by N1-DnsSpm, and N1-DnsSpm could easily permeate channels containing this mutation. The results suggest that at least parts of the M1 and M3 segments contribute to the pore or vestibule of the NMDA channel and that a tryptophan in M2 (W607 in NR2B) may contribute to the narrow constriction of the pore.     

Pore stoichiometry of a voltage-gated chloride channel

Ion channels allow ions to pass through cell membranes by forming aqueous permeation pathways (pores). In contrast to most known ion channels, which have single pores, a chloride channel belonging to the CIC family (Torpedo CIC-0) has functional features that suggest that it has a unique 'double-barrelled' architecture in which each of two subunits forms an independent pore. This model is based on single-channel recordings of CIC-0 that has two equally spaced and independently gated conductance states. Other CIC isoforms do not behave in this way, raising doubts about the applicability of the model to all CIC channels. Here we determine the pore stoichiometry of another CIC isoform, human CIC-1, by chemically modifying cysteines that have been substituted for other amino acids located within the CIC ion-selectivity filter. The CIC-1 channel can be rendered completely susceptible to block by methanethiosulphonate reagents when only one of the two subunits contains substituted cysteines. Thiol side chains placed at corresponding positions in both subunits can form intersubunit disulphide bridges and coordinate Cd2+, indicating that the pore-forming regions from each subunit line the same conduction pathway. We conclude that human CIC-1 has a single functional pore.     

Advanced pain & symptom management (a case study-workshop discussion)

Coexpression of the rat beta 1 subunit with rat brain and skeletal muscle sodium channel alpha subunits in Xenopus oocytes normalizes currents by accelerating sodium current decay kinetics, shifting steady state availability relationships, and accelerating recovery from inactivation. Unlike brain and skeletal muscle, the heart alpha subunit expressed without beta 1 has native-like decay kinetics in oocytes. Messenger RNA for beta 1 has been found in heart, but whether and how it affects cardiac sodium channel function are unclear. We studied coexpression of human heart alpha subunit with beta 1 in Xenopus oocytes using two microelectrode voltage-clamp and macropatch techniques. Coexpression with beta 1 caused a significant positive shift of 3-7 mV in the midpoint of the steady state inactivation relationship but did not affect single-channel conductance, activation, current decay, or recovery from inactivation. Sensitivity to lidocaine block, however, was decreased for both resting state block (Kd = 0.5-1.3 mM) and phasic block in response to pulse trains, but inactivated state block was not affected (Kd = approximately 10 microM). Coexpression with beta 1 increased the rate of recovery from lidocaine block, which accounted for the major part of the observed differences in tonic and phasic block. A beta 1 construct with the cytoplasmic tail removed also produced these effects, demonstrating that the beta 1 cytoplasmic tail was not involved in altering lidocaine block. We conclude that the beta 1 subunit is capable of affecting function of the cardiac sodium channel in oocytes by decreasing tonic and phasic lidocaine block with small effects on gating.     

Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity

Haloperidol and ifenprodil are N-methyl-D-aspartate (NMDA) receptor (NR) antagonists with preference for the NR1/NR2B subunit combination. Previous investigations utilizing 125I-MK801 binding assays with recombinant receptors distinguished certain structural determinants on the NR2B subunit for these two drugs, with glutamate 201 being critical for haloperidol sensitivity and arginine 337 being important for ifenprodil block. Other studies, however, suggested that these two sites pharmacologically overlap. In an attempt to resolve these discrepancies, we have characterized the actions of haloperidol and CP101,606, an ifenprodil analog, on the single-channel properties of NR1/NR2B(E201R) receptors transiently expressed in Chinese hamster ovary cells, because receptors formed by NR1/NR2B(R337K) appear to be nonfunctional. Haloperidol (10 microM) inhibited wild-type NR1/NR2B channels by decreasing the frequency of channel opening, whereas CP101,606 (0.5 microM) antagonized NR1/NR2B channel activity by decreasing both the open dwell time and the frequency of channel opening. The inhibitory actions of both drugs were virtually absent in the mutant NR1/NR2B(E201R) receptors. These results suggest that glutamate 201 is critical for both haloperidol and CP101,606 inhibition, thus demonstrating common features in the action of these two antagonists.     

Molecular determinants of KATP channel inhibition by ATP

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.     

Molecular biology and electrophysiology of calcium-activated potassium channels from lens epithelium

PURPOSE: We structurally and functionally characterized the alpha and beta subunits of the human lens epithelium Ca(++)-activated potassium channel (BK). METHODS: The two subunits were sequenced following RT-PCR with multiple primer pairs. The subunits were cloned using a PCR approach and were expressed in tsA-201 cells for patch clamp recording. Green fluorescence protein-channel subunit fusion proteins were characterized by patch clamping and were imaged by fluorescence microscopy. RESULTS: Alpha subunits alone make a large single-channel conductance, potassium-selective channel with modest Ca++ sensitivity. Beta subunits alone make no channel but, when coexpressed with the alpha subunit, make a channel with increased Ca++ sensitivity, although still less than for natural channels of this type. GFP-BK subunit fusion proteins continue to function and result in a fluorescing channel, which can be localized by fluorescence microscopy. The alpha subunit codes for a "minimal" BK channel in that none of its potential alternative splicing sites contains an "extra" exon. CONCLUSIONS: The Ca(++)-activated potassium channel known as BK has the nucleotide sequences of its alpha and beta subunits represented in messenger RNA of cultured human lens epithelium. It is the first identified channel, to date, which imparts internal Ca++ dependence to lens epithelial potassium conductance.     

Intrinsic dynamics and mechanosensory modulation of non-nutritive sucking in human infants

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (epsilon or gamma), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.     

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure

A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding alpha subunit of the inhibitory glycine receptor forms anion-selective channels in phospholipid bilayers. The most frequent events show single-channel conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau o, in the millisecond time range. These properties match those of authentic glycine receptors studied in inside-out patches of cultured rat spinal cord neurons, namely gamma = 27 pS and gamma = 45 pS, and tau o in the millisecond time range. The channel activity of M2GlyR is sequence-specific: 1) a synthetic peptide with the sequence of putative transmembrane segment M1 (M1GlyR), not considered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays distinct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2GlyR) forms cation-selective channels. Further, a four-helix bundle protein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gamma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, but not by an analog of the local anesthetic lidocaine (QX-222), a cation channel blocker. Observed single-channel properties suggest that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M2 could confer anion specificity to the receptor channel.     

Blocking mechanisms of ketamine and its enantiomers in enzymatically demyelinated peripheral nerve as revealed by single-channel experiments

BACKGROUND: Ketamine shows, besides its general anesthetic effect, a local anesthetic-like action that is due to blocking of peripheral nerve sodium currents. In this study, the stereoselectivity of the blocking effects of the ketamine enantiomers S(+) and R(-) was investigated in sodium and potassium channels in peripheral nerve membranes. METHODS: Ion channel blockade of ketamine was investigated in enzymatically dissociated Xenopus sciatic nerves in multiple-channel and in single-channel outside-out patches. RESULTS: Concentration-effect curves for the Na+ peak current revealed half-maximal inhibiting concentrations (IC50) of 347 microM and 291 microM for S(+) and R(-) ketamine, respectively. The potential-dependent K+ current was less sensitive than the Na+ current with IC50 values of 982 microM and 942 microM. The most sensitive ion channel was the flickering background K+ channel, with IC50 values of 168 microM and 146 microM for S(+) and R(-) ketamine. Competition experiments suggest one binding site at the flicker K+ channel, with specific binding affinities for each of the enantiomers. For the Na+ channel, the block was weaker in acidic (pH = 6.6) than in neutral (pH = 7.4) and basic (pH = 8.2) solutions; for the flicker K+ channel, the block was weaker in acidic and stronger in basic solutions. CONCLUSIONS: Ketamine blockade of sodium and potassium channels in peripheral nerve membranes shows no stereoselectivity except for the flicker K+ channel, which showed a very weak stereoselectivity in favor of the R(-) form. This potential-insensitive flicker K+ channel may contribute to the resting potential. Block of this channel and subsequent depolarization of the resting membrane potential leads, besides to direct Na+ channel block, to inexcitability via Na+ channel inactivation.     

Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor-trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.     

Evaluation of fine needle capillary sampling in superficial and deep-seated lesions. An analysis of 670 cases

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.     

Clustered distribution of calcium sensitivities: an indication of hetero-tetrameric gating components in Ca2+-activated K+ channels reconstituted from avian nasal gland cells

Calcium-activated potassium channels (maxi K+ channels) isolated from avian nasal salt gland cells were reconstituted into lipid bilayers and characterized. The 266 pS channel is blocked discretely by charybdotoxin from the external solution at nanomolar concentrations and by Ba2+ from the cytosolic side at micromolar concentrations. Fast tetraethylammonium (TEA) block is seen as apparent reductions in amplitude of the unitary currents. From the extent of the reductions, TEA binding affinity was calculated to be 0.16 mM from the external solution and 37 mm from internal solution. The overall channel properties conform to those of maxi K+ channels in other epithelial tissues. The calcium sensitivity of the channel was found to be variable from channel to channel, extending over a wide range of concentrations from 1 to 1,000 microM. Examination of the pooled calcium titration curves, revealed that these curves are grouped into five clusters, and the probability distribution of the clusters matches a binomial distribution. The Hill coefficient derived from the titration curves varies from 1 to 5 and is linearly correlated to calcium binding with a slope of 1 per 10-fold change in Kd. Clustered titration curves with such a characteristic suggest that the gating components and the calcium binding sites of the maxi K+ channels in the avian nasal gland are hetero-tetrameric and may result from random mixing of two distinct subunits possessing high and low calcium sensitivities, respectively.     

Maxi-K+ channel in plasma membrane of basal cells dissociated from the stria vascularis of gerbils

The plasma membrane of isolated strial basal cells has been probed for conductive pathways by the patch-clamp single-channel recording technique. Maxi-K+ channels were identified in 28 excised patches (i.e., 29%) out of 95, and these active patches each contained an average of 2.4 channel activities. In the cell-attached mode, activity of the maxi-K+ channel was also observed. Properties of the maxi-K+ channel thus revealed include: (1) linear I-V relations with 150 mM K+ on both sides of the membrane, (2) a unit conductance of 246.2 +/- 4.0 pS (n = 14). (3) Ca2- sensitivity, (4) activation by membrane depolarization. (5) a complete block by Ba2- (2 mM) from either side of the membrane. (6) a flickering block by quinine (0.1 mM) or verapamil (0.1 mM) from either side of the membrane, and (7) a complete block by tetraethylammonium (1 mM) from the outside only. The maxi-K+ channel may play a role in the generation of endocochlear potentials.     

A quantitative immunoassay utilizing Escherichia coli cells possessing surface-expressed single chain Fv molecules

A facile, quantitative immunoassay is described that utilizes Escherichia coli (E. coli) bacteria expressing single chain Fv (scFv) antibody fragments attached to the cell surface. A Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity (Kd = 2.2 x 10(-9) M) and that there are 60,000 scFv molecules per cell. The cells are used as the antibody reagent in the assay, and, following incubation with analyte, simple centrifugation is used to separate the antibody-bound from unbound analyte. The immunoassay is rapid and accurate down to the nanomolar level. In addition, a variety of detection strategies can be used, and the immunoassay is not adversely affected by the presence of animal serum. A key advantage of the new immunoassay is that the antibody reagent can be inexpensively produced in a "ready to use" form by simply growing cultures of the bacteria.     

Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor

We have applied the substituted-cysteine-accessibility method (SCAM) to the M2 segment and the M1-M2 loop of the acetylcholine (ACh) receptor beta subunit. Each residue from beta P248 to beta D273 was mutated one at a time to Cys, and the mutant beta subunits were expressed together with wild-type alpha, beta, and delta subunits in Xenopus oocytes. For each of the mutants, the ACh-induced current was near wild-type. The accessibility of the substituted Cys was inferred from the irreversible inhibition or potentiation of ACh-induced current by methanethiosulfonate (MTS) derivatives added extracellularly. Inhibition by MTSethylammonium of beta G255C, in the narrow part of the channel, was mainly due to a reduction in the single-channel conductance. Conversely, potentiation by MTSethylammonium of beta V266C, in a wider part of the channel, was mainly due to an increase in channel open-time. Two substituted Cys at the intracellular end of M2 and three at the extracellular end were accessible to MTSethylammonium in the absence of ACh. Three additional Cys in the middle of M2 and three in the M1-M2 loop were accessible in the presence of ACh. In the presence of ACh, the secondary structure of beta M2 is alpha-helical from beta G255 to beta V266 and extended from beta L268 to beta D273. The accessible residues in beta M2 are remarkably hydrophobic, while the accessible residues in the M1-M2 loop are charged. beta M2, like alpha M2, alpha M1, and beta M1, undergoes widespread structural changes concomitant with gating, but the gate itself is close to the intracellular end of the channel. Many aligned residues in the M2 segments of alpha and beta are not identically accessible, indicating that the two subunits contribute differently to the channel lining.     

Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 > 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.     

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.     

The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels

BACKGROUND: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.     

Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on both the [3H]ryanodine binding and single-channel measurements were dose-dependent, exhibiting an IC50 of approximately 2.4 mM in binding experiments. Scatchard analysis demonstrated that GSH decreased the binding affinity of ryanodine for its receptor (increased Kd) and lowered the maximal binding occupancy (Bmax). In addition, GSH did not modify the Ca2+ dependence of [3H]ryanodine binding. In single-channel experiments, GSH (5-10 mM), added to the cis side of the bilayer lipid membrane, lowered the open probability (Po) of a Ca2+ (50 microM)-stimulated Ca2+ channel without modifying the single-channel conductance. Subsequent perfusion of the cis chamber with an identical buffer, containing 50 microM Ca2+ without GSH, re-established Ca2+-stimulated channel gating. GSH did not inhibit channel activity when added to the trans side of the bilayer lipid membrane. Similar to GSH, the thiol-reducing agents dithiothreitol and beta-mercaptoethanol also inhibited high affinity [3H]ryanodine binding to sarcoplasmic reticulum membranes. In contrast to GSH, glutathione disulfide (GSSG) was a potent stimulator of high affinity [3H]ryanodine binding and it also stimulated the activity of the reconstituted single Ca2+ release channel. These results provide direct evidence that glutathione interacts with reactive thiols associated with the Ca2+ release channel/ryanodine receptor complex, which are located on the cytoplasmic face of the SR, and support previous observations (Liu, G, Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200) that reactive thiols may be involved in the gating of the Ca2+ release channel.