Preparation and Characterization of 4-Acetamido-4'-Isothiocyanostilbene-2,2'-Disulfonic Acid (Sits) And Related Stilbene Disulfonates

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonicacid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 'H and ¹³C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfon-ate (DIDS) also decomposed in solution, while disodium 4,4'-dinilrostilbene-2, 2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.     

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS; Ki = 10 microM)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4'-aminostilbene 2,2'-disulfonate (BADS; Ki = 2 microM), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4 degrees C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4 degrees C, the weakly bound form was slowly converted to the strongly bound form. At 37 degrees C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37 degrees C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37 degrees C.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate

The permeabilities of sarcoplasmic reticulum vesicle membrane for various ions and neutral molecules were measured by following the change in light scattering intensity due to the osmotic volume change of the vesicles. 4-Acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS), which is a potent inhibitor for the anion permeability of red blood cells membrane, inhibited the permeability of sarcoplasmic reticulum for anions such as Cl-, Pi and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na+, K+, choline and glycerol. Binding of 5 mumol SITS/g protein was necessary for the inhibition of anion permeability. These results suggest the existence of a similar anion transport system in sarcoplasmic reticulum membrane as revealed in red blood cell membrane.     

Effect of disulfonic stilbene anion-channel blockers on the guinea-pig myocardium

The role of anions in the maintenance of tension in electrically driven left atria isolated from guinea pigs has been examined. The disulfonic stilbene anion-channel blockers SITS (4-acetamido-4'-isothiocyanostilbene 2'-disulfonate) and DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) decreased the contractile force developed in a time- and concentration-dependent manner. As in the red cell anion channel, DIDS was more potent than SITS, but the maximal inhibition of tension produced by N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) was considerably lower than the near maximal inhibition produced by SITS and DIDS. The inhibition by SITS and DIDS was irreversible, suggesting a covalent interaction, and could not be overcome by increasing the calcium concentration or the frequency of stimulation. Consistent with a requirement for chloride anion, substitution of chloride and bicarbonate by the impermeant anion gluconate did not support contraction, while only partial tension was maintained with the lipophilic anions acetate and thiocyanate. Incubation of atria with 400 microM SITS blocked both 36Cl and 45Ca uptake to a similar extent, whereas the efflux of both these ions was not affected by incubation of the atria with SITS. The blockade by disulfonic stilbene anion-channel blockers of the contraction of the guinea pig myocardium may result from impairment of excitation-contraction coupling.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Probing the active site of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria: carbodiimide-catalyzed acylation of a functional lysine residue.

Upon modification of the reconstituted aspartate/glutamate carrier by various amino acid-reactive chemicals a functional lysine residue at the exofacial binding site was identified. The inactivation of transport function by the lysine-specific reagents pyridoxal phosphate (PLP, IC50 400 microM) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS, IC50 300 microM) could specifically be suppressed by the substrates aspartate and glutamate; a 50% substrate protection was observed at half-saturation of the external binding site. The same held true for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, IC50 500 microM) and diethyl pyrocarbonate (DEPC, IC50 20 microM), two reagents known to modify carboxylic or histidinyl side-chains, respectively. EDC, however, turned out to catalyze an acylation of the active site lysine by activating carboxyls that had to be present in the incubation medium. This special mechanism, which was proven by protein labelling using EDC/[14C]succinate, necessitates a lysine side-chain of high reactivity and low pK, since the modification had to occur at pH less than or equal to 6.5, i.e. not too far from the pK of the carboxyl to be activated. All reagents applied, additionally including 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, IC50 10 microM), were effective at this pH. Competition experiments revealed interaction of EDC, PLP, SITS and probably DIDS at the same active site lysine. For DEPC a lysine modification could not be ruled out. Yet, a model comprising a histidine juxtaposed to the lysine seems to be appropriate.     

Cyclic AMP-stimulated chloride fluxes in dialyzed barnacle muscle fibers

Unidirectional chloride efflux and influx were studied in giant barnacle muscle fibers that were internally dialyzed. When cyclic 3'5'- adenosine monophosphate (cAMP) was included in the dialysis fluid, both unidirectional fluxes were stimulated by about the same amount. This stimulation was not associated with measurable changes either in membrane electrical conductance or with net movements of chloride. The stimulation required the trans-side presence of chloride. The stimulated flux was inhibited by the sulfonic acid stilbene derivatives 4-acetamido-4'-isothiocyanostilbene-2',2'-disulfonate (SITS) and 4,4'- diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by furosemide. When cAMP was presented in high concentrations (10-5 M), the effect on chloride fluxes was characterized by a desensitization phenomenon. This desensitization was not the result of an increased amount of phosphodiesterase activity, but may be related to ATP and/or intracellular calcium levels. These results further support the hypothesis that the barnacle sarcolemma possesses a specialized chloride transport mechanism that largely engages in Cl-Cl exchange under conditions of normal intracellular pH.     

Ion transport by lasalocid A across red-blood-cell membranes. A multinuclear NMR study

Na+ and K+ fluxes mediated by lasalocid A across erythrocyte membranes have been determined from 23Na-NMR peak areas and chemical shifts, respectively. In similar experiments, Cl- transport has been monitored by NMR signal intensities. Taking into account the external pH variations, the results are readily explainable in terms of charge-balance conservation. The effect of disodium 4,4'-diisothiocyanostilbene-2,2'-disulfonate, an anion-exchange inhibitor, has also been studied.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Affinity labeling of erythrocyte band 3 protein with pyridoxal 5-phosphate. Involvement of the 35,000-dalton fragment in anion transport

Transport of pyridoxal 5-phosphate (PLP) into erythrocytes was inhibited by inhibitors of anion transport including stilbene disulfonate compounds, indicating that it is mediated by Band 3 protein. When erythrocytes were treated with PLP and large amounts of free lysine and NaBH4, two membrane-spanning fragments of Band 3 (Mr = 17,000 and 35,000) were specifically labeled. When the cells were pretreated with 4,4'-dinitrostilbene 2,2'-disulfonate, the labeling in the 35,000-dalton fragment was inhibited. Erythrocytes labeled by PLP in both the 17,000- and 35,000-dalton fragments transported PLP at a decreased rate, whereas the cells labeled in only the 17,000-dalton fragment had essentially the same transport activity as the control when 4,4'-dinitrostilbene 2,2'-disulfonate was removed. The extent of inhibition of transport of inorganic phosphate in the labeled cells was similar to that of PLP. The results indicate that the 35,000-dalton fragment participates in the anion transport of the cell membrane.     

Identification, purification, and characterization of a stilbenedisulfonate binding glycoprotein from canine kidney brush border membranes. A candidate for a renal anion exchanger

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.     

Comparison of murine band 3 protein-mediated Cl- transport as measured in mouse red blood cells and in oocytes of Xenopus laevis

Murine band 3 protein was expressed in oocytes of Xenopus laevis after microinjection of the mRNA from the spleens of anemic mice. The 36Cl- efflux from the oocytes was compared with the chloride fluxes measured in murine red cells. In both oocytes and red cells, the band 3-mediated chloride transport showed the following features: the selective inhibitor of band 3-mediated anion transport, 4,4'-dinitrostilbene-2,2'-disulfonate exerts its effects only when applied to the outside and not when applied to the inside of the membrane. The K1/2 for inhibition by external 4,4'-dinitrostilbene-2,2'-disulfonate was of the order of 1.5 to 2.0 mumol/l. Flufenamate and persantine also produce similar inhibitory effects. Decreasing the pH from 7.4 to 6.0 leads to some inhibition. It is concluded that essential features of the mode of action of murine erythroid band 3 protein in the plasma membrane of the oocyte are similar to the mode of action in the bilayer of the red blood cell of the mouse.     

The use of triplet-sensitized photochromism phenomenon for the study of dynamic interaction of membrane proteins

Possibility of registration of protein interactions in the membranes was demonstrated. The membrane preparation of Na+, K+ ATPase was used in the investigations. The Na+, K+ ATPase was bound with 4-acetoamido-4'-isothiocyanatostilbene-2,2' disullfonic acid (SITS) and erythrosinisothiocyanate (ERITC). The label/Na+,K+ATPase (M/M) ratio was equal to 1:1 for SITS and changed from 1:1 to 1:5 for ERITC. The cis-trans isomerization of SITS was initiated by triplet-triplet energy transfer from light excited ERITC to SITS. The kinetics of isomerization was recorded by the SITS fluorescence measurements. The rate constant of triplet-triplet energy transfer (kT) from ERITC to cis isomer of SITS, (3 divided by 7) X 10(3) M-1 s-1 was determined at 25 degrees C. The kT value of the energy transfer between loose molecules of erythrosine and SITS in buffer solution equaled to 7 X 10(7) M-1 s-1. This drop of kT in the membrane at 10(4) reflected the decrease in the frequency of label collisions caused by the increase in the media viscosity and steric hindrances.     

Hypertonic cryohemolysis: Ionophore and pH effects

Human erythrocytes suspended at 37 degrees C in hypertonic solution of either electrolytes or nonelectrolytes undergo hemolysis when the temperature is lowered toward 0 degrees C (Green, F.A., Jung, C.Y. 1977 J. Membrane Biol. 33:249). In the present studies this hypertonic cryohemolysis was profoundly affected by the pH of incubation, and was completely abolished at ph 5. In hypertonic NaCl, there was an apparent pH optimum at 6--6.5. In hypertonic sucrose, on the other hand, hemolysis increased progressively with increasing pH between 6 and 9. Amphotericin B inhibited hypertonic cryohemolysis in NaCl or KCl solution. No inhibiting effect of amphotericin B was observed when hypertonicity was due to sodium sulfate or sucrose. Valinomycin also inhibited hypertonic cryohemolysis in KCl, but did not affect the process in NaCl or sucrose solution. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) and phloretin interfered with this valinomycin effect, whereas phlorizin did not. These results indicate that dissipation of an osmotic gradient across membranes may be responsible for the inhibition of the hemolysis by these inophores. Iso-osmotic cell shrinkage induced by valinomycin in 150 mM NaCl solution did not result in cryohemolysis.     

Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion

Stilbene disulfonic acids inhibit surfactant secretion from lung epithelial type II cells by an undefined mechanism, and inhibit CD4 mediated cell-cell fusion. We have previously shown that lung synexin promotes in vitro fusion of lamellar bodies and plasma membranes, an obligatory process for surfactant secretion. This study investigates the effect of stilbene disulfonic acids, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMDS), on synexin-mediated liposome aggregation and fusion. Structurally, these three stilbene compounds differ in the number of isothiocyano groups present (DIDS = 2, SITS = 1, and AMDS = 0). At 10 μg synexin/ml, DIDS and SITS inhibited synexin-mediated liposome aggregation with an EC₅₀ of 3.5 μM and 148 μM, respectively. In comparison, AMDS was least inhibitory (EC₅₀ > 1 mM). Thus, the inhibitory potency (DIDS > SITS > AMDS) was partly dependent upon the number of isothiocyano groups. The EC₅₀ was also dependent on synexin concentration. Stilbene disulfonic acids were also inhibitory for arachidonic acid-enhanced synexin-mediated liposome fusion. The EC₅₀ for DIDS and SITS for fusion were similar to that for liposome aggregation. Ca²⁺-induced synexin polymerization, measured by 90° light scattering, was increased by DIDS, suggesting binding of stilbene disulfonic acids to synexin. The binding of DIDS to synexin was dependent on the molar ratio of synexin to DIDS. These results indicate that stilbene disulfonic acids interact directly with synexin to inhibit membrane aggregation and fusion. Our results suggest that such inhibition of synexin activity may contribute towards inhibition of surfactant secretion by DIDS, and support a physiological role for synexin in lung surfactant secretion.     

Permeation of the erythrocyte stroma by superoxide radical.

Superoxide anion, generated by xanthine oxidase within vesicles formed from washed erythrocyte ghosts, crosses the vesicle membrane to reduce cytochrome c in the medium (Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem, 253, 1838-1845). To determine whether O2- could travel through the membrane in the "channel" for the exchange of stable anions, the effects of two specific inhibitors of anion exchange, 4-acetamido-4'-isothiocyano-2,2' disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS), on the escape of O2- from vesicles were studied. The reduction of external cytochrome c, caused by O2- produced by the enzymic turnover of internal xanthine oxidase, was 85 to 90% inhibited by SITS and DIDS. If SITS impeded the egress of O2- from vesicles, it should enhance the internal effects of O2- and antagonize the inhibition of these effects by external superoxide dismutase. External superoxide dismutase inhibited the lysis of vesicles containing xanthine oxidase. SITS (60 micron) partially reversed this inhibition. It appears that O2- can cross the membrane of the erythrocyte in the anion channel.     

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.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

Purification of a stilbene sensitive chloride channel and reconstitution of chloride conductivity into phospholipid vesicles.

A protein conferring passive chloride permeability was isolated from a N-octylglucoside solubilized extract of partially purified H(+)-transporting osteoclast cell membranes. Purification was achieved by binding of solubilized protein to an amine-linked 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) Sepharose 4B column and elution with 50 mM KCl. A major protein, with MR = 60 kD on 10% SDS-PAGE, was obtained, which was further purified to homogeneity by HPLC gel filtration. This protein introduced 36Cl- permeability when reconstituted in phospholipid membranes by equilibrium dialysis. The Cl- transport recovered in reconstituted membranes retained sensitivity to DIDS confirming the identity of the isolated protein as a stilbene-sensitive chloride channel.     

Voltage-dependent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives

When giant axons of squid, Sepioteuthis, were bathed in a 100 mM Ca-salt solution containing tetrodotoxin (TTX) and internally perfused with a solution of 100 mM tetraethylammonium-salt (TEA-salt) or tetramethylammonium-salt (TMA-salt), the membrane potential was found to become sensitive to anions, especially Cl-. Membrane currents recorded from those axons showed practically no time-dependent properties, but they had a strong voltage-dependent characteristic, i.e., outward rectification. Cl- had a strong effect upon the voltage-dependent membrane currents. The nonlinear property of the currents was almost completely suppressed by some disulfonic stilbene derivatives applied intracellularly, such as 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are blockers of chloride transport. On the basis of these experimental results, it is concluded that a voltage-dependent chloride-permeable channel exists in the squid axon membrane. The chloride permeability (PCl) is a function of voltage, and its value at the resting membrane (Em = -60 mV) is calculated, using the Goldman-Hodgkin-Katz equation, to be 3.0 X 10(-7) cm/s.