Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle

Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2'-disulfonic acid (DBDS), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)] was investigated in planar bilayers using SO4(2-) as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1-3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 microM for DBDS and 3.4 and 55 microM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14-24 A and that the pore constriction is approximately 10 A in diameter.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

A systematic study was made of the action of 4-acet-amido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer 45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS, Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton.     

Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum

Ca2+ channels of isolated sarcoplasmic reticulum were incorporated into a planar lipid bilayer and their pharmacological properties were studied. The results show that the channel is a Ca2+-induced Ca2+ release channel like that observed in skinned muscle fibers and isolated vesicles. (i) The open channel probability was increased by the addition of micromolar amounts of Ca2+ to the cis (myoplasmic) side and further increased by millimolar ATP. (ii) The channel was closed by millimolar Mg2+ and micromolar ruthenium red. We found that two disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), when added to the cis side open the channel and lock it irreversibly at open without changing the single channel conductance. Ca2+ efflux from SR vesicles was also enhanced by SITS and DIDS, as monitored by a tracer assay. Further, Ag+ activated the channel transiently. These results suggest that certain amino and SH residues play important roles in gating the Ca2+ channel.     

Stilbene disulfonic acids. CD4 antagonists that block human immunodeficiency virus type-1 growth at multiple stages of the virus life cycle

The stilbene disulfonic acids 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid and, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid bound the variable-1 immunoglobulin-like domain of CD4 on JM cells. The interaction blocked the binding of the anti-CD4 monoclonal antibody OKT4A and the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1). DIDS inhibited the acute infection of CD4+ cells by HIV-1 with a potency (IC50 approximately 30 microM) similar to that which blocked gp120 binding (IC50 approximately 20 microM) to the cellular antigen. Pretreating uninfected CD4+ C8166 cells with DIDS blocked their fusion with chronically infected gp120+ cells. DIDS covalently and selectively modified lysine 90 of soluble CD4 and abolished the gp120-binding and antiviral properties of the recombinant protein. When added to cells productively infected with HIV-1, DIDS blocked virus growth and cleared cultures of syncytia without inhibiting cellular proliferation. The stilbene disulfonic acids are a novel class of site-specific CD4 antagonists that block multiple CD4-dependent events associated with acute and established HIV-1 infections.     

Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.     

DIDS modifies the conductance,gating, and inactivation mechanisms of the cardiac ryanodine receptor

The effects of the covalent modifier of amino groups, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on the single-channel properties of purified sheep cardiac ryanodine receptors (RyR) incorporated into planar phospholipid bilayers were investigated. DIDS increased single-channel conductance and open probability (P(o)) and induced unique modifications to the voltage-dependence of gating. The effects of DIDS on conduction and gating were irreversible within the time scale of the experiments, and both effects were dependent on the permeant ion. DIDS induced a greater increase in conductance with Ca(2+) (20%) compared with K(+) (8%) as the permeant ion. After modification by DIDS, all channels could be rapidly inactivated in a voltage-dependent manner. The open probability of the DIDS-modified channel decreased with increasing positive or negative transmembrane potentials; however, inactivation was only observed at negative potentials. Our results demonstrate that inactivation of RyR channels is dependent on the ligand activating the channel, and this will have consequences for the control and termination of sarcoplasmic reticulum Ca(2+) release in cardiac cells.     

Effects of 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis

Cell viability requires the perfect functioning of the processes controlling ATP and Ca(2+) homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with a disruption of ATP and Ca(2+) homeostasis. This study shows that 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits Trypanosoma cruzi epimastigote cell growth. This thiol-reagent thiocyanate derivative was able to inhibit two ecto-enzymes present in this parasite. The ecto-ATPase and ecto-phosphatase activities were inhibited in a dose-dependent manner (K(i)=47.7 and 472.5 microM, respectively), but the 5'nucleotidase and 3'nucleotidase activities were not. DIDS uptake was approached by fluorescence microscopy. Pulse-chase experiments revealed the DIDS accumulation in compartments, presumably endocytic, in the posterior region of epimastigotes. In addition, we show that the T. cruzi mitochondria studied in permeabilized cells are able to accumulate and retain medium Ca(2+) in the absence of DIDS. However, in the presence of increasing concentrations of DIDS (50-200 microM), Ca(2+) transport was inhibited in a dose-dependent manner. DIDS also caused a disruption of the mitochondrial membrane potential, in the same concentration range, thus explaining its effect on Ca(2+) uptake. The presence of EGTA prevented the elimination of the mitochondrial membrane potential (DeltaPsi), supporting previous data suggesting that the binding of Ca(2+) to the mitochondrial membrane exposes buried thiols to react with DIDS. This thiocyanate derivative was also able to inhibit Ca(2+) uptake by the endoplasmic reticulum in a dose-dependent manner. Taken together, the data presented here provide further insights into the mechanisms underlying the antiproliferative actions of DIDS in T. cruzi.     

Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange

Treatment of whole erythrocytes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) results in inhibition of ATP and phosphate-dependent Ca2+ transport in subsequently prepared inside-out vesicles (IOV). Accumulation of phosphate into IOV in the presence of Ca2+ is virtually abolished by prior DIDS treatment, consistent with the presumed inhibition of the band III anion-exchange protein by this agent. No inhibition of Ca2+-activatable ATP hydrolysis is observed following DIDS treatment when open membranes are used to prevent development of ion gradients. This indicates that DIDS does not affect the inherent ATPase activity of the calcium pump (Waisman, D. M., Smallwood, J., Lafreniere, D., and Rasmussen, H. (1982) FEBS Lett. 145, 337-340). In IOV prepared from untreated cells, ATP-dependent Ca2+ uptake is stimulated by phosphate, sulfate, or chloride. Rates of Ca2+ uptake into DIDS-IOV are not increased by these anions. Lipid-permeable organic acids such as acetate, however, do promote Ca2+ transport in DIDS-IOV. Lipophilic anions incapable of transporting protons into the vesicle interior (nitrate and thiocyanate) support sustained uptake only when the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone is also added. These results support a model of the (Ca2+-Mg2+)-ATPase as a pump exchanging Ca2+ for protons, not transporting Ca2+ alone. Band III protein appears to promote Ca2+ transport in the presence of phosphate, sulfate, or chloride by exchanging external anion for the accumulating OH- (or HCO3-) produced by the calcium pump.     

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid inhibits CD3-T cell antigen receptor-stimulated Ca2+ influx in human T lymphocytes

Stimulation of the CD3-T cell antigen receptor complex on T lymphocytes results in a rapid rise in intracellular calcium from both intra- and extracellular sources. The former is thought to be released from the endoplasmic reticulum in response to inositol trisphosphate, while the latter enters the cells through a membrane potential-sensitive transporter (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590). In this report we show that the stilbene disulfonate, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), inhibited the ability of monoclonal anti-CD3 complex antibodies to stimulate an influx of calcium in the human T lymphocyte cell line, Jurkat. DIDS had no effect on either antibody binding to the receptor or receptor-stimulated phosphatidylinositol turnover. The Ki was approximately 25 microM in the presence of extracellular Cl- and 10 microM when labeling was performed in the absence of Cl-, suggesting that DIDS was competing with Cl- for binding to the cell membrane. The reduced form of DIDS, dihydroDIDS, was only 50% as effective as DIDS itself, and the monoisothiocyanate stilbene, 4-acetamido-4'-isothiocyantostilbene-2,2'-disulfonic acid, was totally ineffective, even to concentrations of 0.750 mM. Removal of extracellular Cl- also inhibited the antibody-stimulated influx of calcium. These data suggest that the function of the CD3-T cell receptor-activated calcium channel/transporter may be dependent on or regulated by extracellular Cl-.     

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.     

Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle

Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.     

Voltage-gated anion channel of the electric organ of Narke japonica incorporated into planar bilayers

Voltage-gated anion channels in vesicles prepared from the electric organ of Narke japonica were studied using two methods. Ionic permeability was measured by the light scattering method, which could be used to measure the ion permeation of whole vesicles but only at a time scale of slower than about 0.1 s. The single channel conductances and permeability ratios for various ions were measured after fusing the vesicles to phospholipid bilayers. Both sets of results coincided, indicating that the anion channels observed with the planar bilayer method are the major route for anion passage in these vesicles. The channels showed anion selectivity and did not allow the permeation of cations such as K+ and choline+. The single channel conductance was 18 pS in 0.1 M Cl-. SCN- inhibited the conductance in a voltage-dependent reversible manner on both sides of a channel. SCN- may bind to the Cl- binding site in a channel and thus block it. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocked a channel on the cis (extracellular) side irreversibly. The number of anion channels per vesicle was estimated to be about 50. It was also shown that all anion channels in the vesicles were open at the very instance of fusion with planar membranes.     

Effect of anion transport blockers on CFTR in the human sweat duct

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.     

Ion conductance pathways in potato tuber (Solanum tuberosum) inner mitochondrial membrane

Single-ion channel activities were measured after reconstitution of potato tuber mitochondrial inner membranes into planar lipid bilayers. In addition to the recently described large-conductance Ca(2+)-activated potassium channel activity (Koszela-Piotrowska et al., 2009), the following mitochondrial ion conductance pathways were recorded: (i) an ATP-regulated potassium channel (mitoK(ATP) channel) activity with a conductance of 164+/-8pS, (ii) a large-conductance Ca(2+)-insensitive iberiotoxin-sensitive potassium channel activity with a conductance of 312 pS+/-23, and (iii) a chloride 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-inhibited channel activity with a conductance of 117 pS+/-4. In isolated non-phosphorylating potato tuber mitochondria, individual and combined potassium channel activities caused significant (up to 14mV) but not collapsing K(+)-influx-induced membrane potential depolarisation. Under phosphorylating conditions, the coupling parameters were unchanged in the presence of high K(+) level, indicating that plant K(+) channels function as energy-dissipating systems that are not able to divert energy from oxidative phosphorylation. A potato tuber K(+) channel that is ATP-, 5-hydroxydecanonic acid-, glybenclamide-inhibited and diazoxide-stimulated caused low cation flux, modestly decreasing membrane potential (up to a few mV) and increasing respiration in non-phosphorylating mitochondria. Immunological analysis with antibodies raised against the mammalian plasma membrane ATP-regulated K(+) channel identified a pore-forming subunit of the Kir-like family in potato tuber mitochondrial inner membrane. These results suggest that a mitoK(ATP) channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.     

Inhibition and derivatization of the renal Na,K-ATPase by dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate

Treatment of purified renal Na,K-ATPase with dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) produces both reversible and irreversible inhibition of the enzyme activity. The reversible inhibition is unaffected by the presence of saturating concentrations of the sodium pump ligands Na+,K+, Mg2+, and ATP, while the inactivation is prevented by either ATP or K+. The kinetics of protection against inactivation indicate that K+ binds to two sites on the enzyme with very different affinities. Na+ ions with high affinity facilitate the inactivation by H2DIDS and prevent the protective effect of K+ ions. The H2DIDS-inactivated enzyme no longer exhibits a high-affinity nucleotide binding site, and the covalent binding of fluorescein isothiocyanate is also greatly reduced, but phosphorylation by Pi is unaffected. The kinetics of inactivation by H2DIDS were first order with respect to time and H2DIDS concentration. The enzyme is completely inactivated by the covalent binding of one H2DIDS molecule at pH 9 per enzyme phosphorylation site, or two H2DIDS molecules at pH 7.2. H2DIDS binds exclusively to the alpha-subunit of the Na,K-ATPase, locking the enzyme in an E2-like conformation. The profile of radioactivity, following trypsinolysis and SDS-PAGE, showed H2DIDS attachment to a 52-kDa fragment which also contains the ATP binding site. These results suggest that H2DIDS treatment modifies a specific conformationally sensitive amino acid residue on the alpha-subunit of the Na,K-ATPase, resulting in the loss of nucleotide binding and enzymatic activity.     

Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates

The anion transporter from human red blood cells, band 3, has been expressed in Xenopus laevis frog oocytes microinjected with mRNA prepared from the cDNA clone. About 10% of the protein is present at the plasma membrane as determined by immunoprecipitation of covalently bound 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) with anti-DIDS antibody. The expressed band 3 transport chloride at a rate comparable to that in erythrocytes. Transport of chloride is inhibited by stilbene disulfonates, niflumic acid, and dipyridamole at concentrations similar to those that inhibit transport in red blood cells: DIDS and 4,4'-dinitro-2,2'-stilbene disulfonate inhibit chloride uptake with Kiapp of 34 nM and 2.5 microM, respectively. Lysine 539 has been tentatively identified as the site of stilbene disulfonate binding. Site-directed mutagenesis of this lysine to five different amino acids has no effect on transport. Inhibition by stilbene disulfonates or their covalent binding was not affected when Lys-539 was substituted by Gln, Pro, Leu, or His. However, substitution by Ala resulted in weaker inhibition and covalent binding. These results indicate that lysine 539 is not part of the anion transport site and that it is not essential for stilbene disulfonate binding and inhibition.     

Spontaneous oscillation and mechanically induced calcium waves in chondrocytes

The characteristics of spontaneous calcium (Ca(2+)) oscillation and mechanically induced Ca(2+) waves in articular chondrocytes were studied. In some, but not all, chondrocytes in sliced cartilage and primary cultures, we observed spontaneous oscillation of intracellular Ca(2+) that never spread to adjacent cells. In contrast, a mechanical stimulus to a single cell by touching with a glass rod induced an increase of intracellular Ca(2+) that spread to neighboring cells in a wave-like manner, even though there was no physical contact between the cells. This indicated the release of some paracrine factor from the mechanically stimulated cells. Application of ultrasonic vibration also induced an oscillation of intracellular Ca(2+). The application of a uridine 5'-triphosphate (UTP), UTP, induced a transient increase in intracellular Ca(2+) and the release of adenosine 5'-triphosphate (ATP) in cultured chondrocytes. A P2 receptor antagonist (suramin) and blockers of Cl(-) channels, niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), reduced the UTP-induced ATP release. The results indicated that Cl(-) channels were involved in the extracellular release of ATP following mechanical or P2Y receptor stimulation. Thus, ATP stimulation of P2Y receptors elicits an increase in intracellular Ca(2+), triggering further release of ATP from adjacent cells, thereby expanding the Ca(2+) wave in chondrocytes.     

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.