The Mitochondrial Inner Membrane Anion Channel - The Journal of ...

Vol. 262, No. 31, Issue of November 5, pp. 15085-15093,1987 Printed in U.S. A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular’ Biology, Inc

The Mitochondrial Inner Membrane Anion Channel REGULATION BY DIVALENT CATIONS AND PROTONS* (Received for publication, May 14, 1987)

Andrew D. BeavisS and Keith D. Garlid From the Department of Pharmacology, MedicalCollege of Ohio, Toledo, Ohio 43699

be ejected from the matrix by the high membrane potential generated by respiration. In view of these considerations it is interesting that certain “nonphysiological” conditions, such as high pH (2-10) and low osmolality (5, l l ) , open up an electrophoretic anion uniport pathway with broad selectivity in the inner membrane of both liver and heart mitochondria. The groups of both Azzone (4, 5) and Brierley (7, 12, 13) have attributed the increase in anion fluxes observed under these extreme conditions to changes in the permeability of the lipid bilayer. On the other hand, Selwyn’s group (10, 14) has proposed that a specific “pH-dependent anionconducting pore” is opened up; however, the existence of an anion uniport protein has not become generally accepted. Possible reasons for this are the facts that it has no obvious function and that itonly appears to be active under extreme conditions. Furthermore, it follows from chemiosmotic theory that if an anion could be transported both electroneutrally and electrophoretically, as is the case for the classic uncouplers (15), futile anion cycling could uncouple oxidative phosphorylation (16). Despite these considerations, as we have discussed elsewhere (17), a well controlled anion uniport pathway in mitochondria could serve a number of useful functions. In this communication we present evidence that an anion uniport pathway can be opened up at neutral pHby depleting mitochondria of divalent cations. We also demonstrate that this pathway has a very broad specificity for anions and that it is regulated by protons. On the basis of these findings, we conclude that divalent depletion and alkaline pH probably A number of carriers have been identified for the transport activate the same transport pathway, which we refer to as of anionic substrates across the inner mitochondrial mem- inner membrane anion channel (17). brane; however, none involved in a major metabolic pathway catalyzes electrophoretic uniport (see Ref. 1 for a review). EXPERIMENTALPROCEDURES Most inner membrane carriers catalyze net transport of the Mitochondrial Preparations--Rat liver mitochondria, isolated by acid or electroneutral exchange of the anion for another anion; differential centrifugation as previously described (18),were resushowever, twocatalyze the electrophoretic exchange of anions. pended to 50 mg of protein/ml in 0.25 M sucrose and stored on ice. The adenine nucleotide translocator catalyzes the exchange “‘+-depleted mitochondria were prepared by adding one part of stock suspension to four parts of pretreatment medium. The resulting of ATP4- for ADP3- andthe aspartate/glutamatecarrier catalyzes the exchange of Asp- for Glu- + H’. Allof these mitochondrial suspension was 110 mosm’ and pH 7.4. Rotenone (1 transport mechanisms are consistent with and provide indi- pg/mg) and A23187 (1 nmol/mg) were added, the suspension was incubated at 25 “C for 2 min to allow K+/H+ antiport to come to rect evidence for the chemiosmotic theory of the coupling of equilibrium, and then was it placed on ice. Two different pretreatment oxidative phosphorylation. This theory predicts that if an media wereused. One contained the K+salts (K+pretreatment anion were transported by electrophoretic uniport its entry medium) and the other NH: salts (NH: pretreatment medium) of into the mitochondrion would be severely limited or it would TES’ (44 mM) and EDTA (5.5 mM). The effect of pretreatment on mitochondrial M8’ and K+ levels was determined by centrifuging of * This work was supported by National Institutes of Health Grants the pretreatment suspension and analyzing both the supernatant and

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as C1-, SO:-, and Fe(CN)$ as well as physiologically important anions such as HCO;, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with lossof matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.

GM 31086 andHL 36573 awarded by the National Institute of General Medical Sciences and the National Heart, Lung, and Blood Institute, United States Public Health Service, Department of Health and Human Services and also by a grantfrom the Northwestern Ohio Chapter of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed.

pellet for M P and K+ by atomic absorption spectroscopy. In both media matrix M$+ was depleted from 38 nmol/mg to about 0.6 nmol/ mg, and in the NH: pretreatment medium mitochondrial K+ was depleted from 145 nmol/mg to about 9 nmol/mg. It should be pointed

The abbreviations used are: mosm, milliosmolal; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid CCCP, carbonyl cyanide m-chlorophenylhydrazone; L.S., light scattering; M2+,divalent cation.

15085

Uniport Anion

15086

out that when the mitochondria were pretreated with A23187 and EDTA to remove matrix M2+,1nmol/mg A23187 was routinely used; however, lower dosesare equally effective, provided sufficient time is allowed for M2+depletion to take place. Doses of A23187 as high as 5-10 nmol/mg are necessary to achieve maximum rates only when A23187 is added directly to theassay medium in which the mitochondria are present at 0.1 mg/ml. Light Scattering (L.S.) Measurements-Uptake of salts and water into the mitochondrial matrix results in matrix swelling and a consequent decrease in the light scattered by the mitochondrial suspension (19, 20). The light scattering variable 3, normalizes reciprocal absorbance (A-’) for mitochondrial concentration, P (mg/ml),

where P, (equals 1 mg/ml) is introduced to make P a scaled dimensionless quantity and a is a machine constant equal to 0.25 with our apparatus (18). Absorbance was measured at 520 nm and sampled 0.01-min intervals with a Brinkmann PC 700 probe colorimeter connected to a Cyborg 41A analog/digital converter. The digitalized signal was passed to anApple IIe computer for conversion to inverse absorbance, real time plotting, and storage. A linear regression routine was used to obtain rates, d@/dt(min”), from light scattering traces. Over the range studied, fi and matrix water ( W,) are both linear with inverse osmolality (18), b P = P 0 + ;

so w, = W b + 9 where is medium osmolality, b (osmolal) and So (nosmol/mg) are the observed slopes of the corresponding equilibrium osmotic curves, and Po and W , (mg/mg) are the corresponding intercepts. W, is matrix water content (sucrose-free water) determined from the distribution of [‘4C]sucroseand 3H20 in parallel gravimetric experiments (18).Since water uptake is much faster than salt transport (18), the rate of salt uptake, J. (nmol/min.mg), is proportional to the L.S. kinetic, @/dt(min”), and the proportionality constant is defined by Equations 2 and 3. For any salt, ignoring the osmotic coefficient of the matrix solutions (21), J

*So =-.-

db’ nb dt

where n is the number of moles of osmotically active particles which make up 1 mol of the salt. Since So is reproducibly found to be 190 nosmol/mg in our laboratory and b is approximately 15 mosm with our equipment, at Q = 110 mosm @So/b is about 1400 nmol/mg. All assay media were made up to be of equal osmolality (110 mosm), e.g. the pH 7.4 KC1 and potassium malonate media contained the K+ salts of c1- (55 mM) or malonate (37 mM), respectively, plus the K+ salts of TES (5 mM), EDTA (0.1 mM), and EGTA (0.1 mM). Rotenone (2 pg/mg) was added just prior to the mitochondria, and the media were maintained a t 25 “C by a circulating water bath. For experiments at other pH values, the concentration of TES was maintained constant and the salt concentration was adjusted slightly to maintain constant osmolality. For assays in ammonium salts the media were identical except that NH4+was substituted for K+. RESULTS

in Mitochondria

1 0.15

r0

1

0

1

1 1’ b

I

I

t (mid

t (mid

2

3

2

3

a

I

FIG. 1. Depletion of matrix divalent cations activates a uniport pathway for C1-. L.S. kinetics exhibited by mitochondria (0.1 mg/ml) suspended in KC1 assay medium are shown. A: trace a, control; trace b, valinomycin (Val, 1 nmol/mg) was added at 1 min; trace c, valinomycin (1nmol/mg) was added at 1 min and A23187 (10 nmol/ mg) wasadded at 2 min. B: trace a, control; trace b, A23187 (10 nmol/ mg) was added at 1min; trace c, A23187 (10 nmol/mg) was added at 1 min, and valinomycin (1 nmol/mg) was added at 2 min. The assay medium is described under “Experimental Procedures.”

depletion of matrix M e (22-25) (see “Appendix”).’ A major contribution by electroneutral Cl-/OH- antiport is, however, ruled out by the findings that A23187 alone only induces a slow rate of swelling (Fig. lB, curve b) and that subsequent addition of valinomycin is necessary to induce rapid swelling (Fig. lB, trace c ) . Although the transport observed in the absence of valinomycin could reflect Cl-/OH- exchange, the increment induced by valinomycin must represent C1- uniport. The Rate of Anion Uniport in NH,’ Salts Is Highly Variable-Experiments similar to those shown in Fig. 1were carried out with ammonium salts using the protonophore CCCP to provide the counterionfor C1- transport (notshown). In these experiments, however, we observed that the rate of swelling was much faster when we added CCCP first than when we added A23187 first. Moreover, even faster rateswere obtained when we added A23187 and CCCP together at zero time. To investigate the possibility that the rateof swelling was related to the lengthof time between depletion of mitochondrial M2+and initiationof swelling, we devised a pretreatment medium containing A23187 and the K+ salts of EDTA and TES to deplete mitochondrial M2+ prior to the assay (see “Experimental Procedures”). When these mitochondriawere transferred toassay mediumcontaining CCCP, to our surprise we still observed very rapid swelling and found that the length of the pretreatmentperiod had virtually no effect on the rate (not shown). Because these mitochondria swelled only very

Divalent Cation Depletion Activates a n Anion UniportPathway-Fig. 1 contains L.S. traces of mitochondria suspended in 110 mosm KC1. In theabsence of A23187 and valinomycin no swelling takes place (Fig. 1,A and B, trace a). Addition of valinomycin (Fig. lA, trace b) induces a slow rate of swelling; Portions of this paper (including “Appendix” and Figs. 10-12) are however, subsequent addition of A23187 to deplete matrix divalent cations induces rapid swelling (Fig. L4, trace c ) . This presented in miniprint at the end of this paper. Miniprint is easily swelling results from net salt influx which could be catalyzed read with the aid of a standardmagnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville by a combination of anion uniport activatedby M2+depletion Pike, Bethesda, MD 20814. Request Document No. 87M-1588, cite and K+ uniport via valinomycin or by a combination of C1-/ the authors, and include a check or money order for $4.80 per set of OH- exchange activated by M2’ depletion and K+/H+ antiportphotocopies. Full size photocopies are also included in the microfilm since it has beenshown that the latterprocess is activatedby edition of the Journal that is available from Waverly Press.

Anion Uniport in Mitochondria slowly in the absenceof CCCP, we were also able to examine the effect of the duration of exposure of M*+-depleted mitochondriatotheassaymediumonthesubsequentrate of CCCP-induced anion transport. Fig. 2 shows the rate of C1transport as a function of thetimeinterval between the addition of the mitochondria to the assay and the addition of the CCCP to the mitochondria.As the interval increases the rate declines dramatically until it reaches about 15% of the maximum rate,at which point itbecomes constant. Note that the addition of both A23187 and CCCP to the pretreatment has only a small effect on the initial rate of C1- transport. Thus, activityof the uniport pathway appears decline to upon exposure of M2+-depleted mitochondria to the assay medium. Variability of Anion Uniport Fluxes in NH: Salts Is Associated with Exchange of Endogenous K+ for Medium NHZWhen we found that the decline inuniportactivity only appears to take place in the assay medium, it occurred to us that thedecline in activity may be related to theexchange of matrixK+ formedium NH:, since it is known that Mg2+ depletion activates the K+/H+ antiporter (22-25). Three experiments were designed to investigate thispossibility. In the first we examined the effect of substituting NH: for K+ in the pretreatment medium. This should permit matrix K+ to be exchanged for NH: and, therefore,if the decline in uniport activity is related to the exchangeof K’ for NH:, the decline in activity should take place prior to transferring the mitochondria to the assay medium. The data inFig. 3 confirm this prediction. When CCCP is included in the medium from zero time, mitochondria pretreated in the K’ medium swell rapidly (truce a ) , whereas those pretreated in the NH: medium swell more slowly (truce b).Moreover, the rate of swelling of mitochondria pretreated in the NH: medium is independent of the time of addition of CCCP (compare traces b and c ) and, furthermore, it is identical to the rate observed when CCCP is added at 1 min to mitochondria pretreated in K+ medium (trace d ) . Thus, consistent with theexchange of K’ for NH: being related to the decline in uniport activity, inclusion of NH: in the pretreatment medium allows the decline in uniport activity to takeplace in the pretreatment mix. In thesecond experiment we looked at the effect of adding nigericin to the assay medium. If the decreasein uniport

0.6

.

JA 0.4

.

0.2

’

‘I

‘L I

?

Y

,

O

-2

-1

0

t (mid

FIG.2. The rate of C1- transport in NH&l is dependent on the timeof addition of the protonophore. The maximum rate of C1- transport (JA,pmol/min.mg) in M2+-depletedmitochondria is plotted uersus the time intervalbetween addition of the mitochondria to the assay and the addition of CCCP (10 nmol/mg) to the mitochondria. The mitochondria were depleted of matrix Mz+ by treatment with A23187 in the K’ pretreatment mix described under “Experimental Procedures.” These mitochondria were then transferred to the assay medium to give a final concentration of 0.1 mg/ ml. The rateof C1- transport was determined from the L.S. kinetics. This procedure and the assay medium are described under “Experimental Procedures.”

15087 a!

i

1

b

0.35

=/’

0

7-/

0.25 CCCP 0.15

0

1

2 t

3

(mid

FIG.3. The effect of pretreatment of mitochondria in K+ salts uers~u) N&+ salts on subsequent swelling in NHaCl. L.S. kinetics exhibited by M2+-depletedmitochondria in NH4CI medium are shown. The mitochondria were M2+-depleted by pretreatment with A23187 in the K+ pretreatment medium (truces a and c) or the described under NH: pretreatment medium (traces b andd)as “Experimental Procedures.” CCCP (10 p ~ was ) added to the assay a t zero time (traces a and b ) or a t 1 min (traces c and d). The rates of anion transport calculated from truces a-d are 530, 135, 140, and 135 nmol of Cl-/min. mg, respectively. The assay medium is described under “Experimental Procedures.”

0.35

.p 0.25

-

t (mid

-

FIG.4. The effect of nigericin on mitochondrial swelling in NH,Cl. L.S. kinetics exhibited by M2+-depleted mitochondria in NH&l medium are shown. The mitochondria (0.1 mg/ml) were added to assay medium which contained A23187 (10 nmol/mg). CCCP (10 nmol/mg) was added at zero time (truces a and b ) or a t 1 min (truces c and d ) . Nigericin (0.5 nmol/mg) was added to truces b and d at zero time. The ratesof anion transport calculated from traces a-d are 520, 77, 76, and 73 nmol of Cl-/min.mg, respectively. The assay medium is described under “Experimental Procedures.’’ activity is related to the exchange of matrix K’ for medium NH:, then the period over which the decline takes place should be the periodrequiredfor equilibration of K+/H+ exchange across the inner membrane. Thus, addition of nigericin should accelerate thedecline in anion uniportactivity. The datashown in Fig. 4 confirm this prediction. When both A23187 and CCCP are included in themedium from zero time very rapid swelling is observed (trace a ) ; however, if nigericin is also included in themedium (trace b ) the maximum rate of swelling is much slower. More importantly, the rateobserved under these conditions is very close to that observed when CCCP is added at 1 min in the absence (truce c ) or presence (trace d ) of nigericin. The lack of effect of nigericin on the rate of swelling induced by the late addition of CCCP suggests that nigericin per se has no effect on the C1- conductance. Thus, like pretreatment in NH: medium,nigericin in the assay mediumsimply appears to accelerate the decline in anion uniport activity. In the third experiment we looked for a change in anion uniport activity in KC1 assay medium. If our explanation for the changesobserves in NH4C1assay medium was correct the rate of swelling shouldnot declineuponexposure of the mitochondria to the assay medium. The data contained in Fig. 5 confirm this prediction. The rateof swelling is in fact slower when valinomycin is added to the assay at zero time

Anion Uniport in Mitochondria

15088 I

0.35

-

’/

;

c /

a

r

b,d j’

,,”

I

0 0.25

0.4

.

:

.’ 7

>: ,’

val

i

-+nig

.I

1

, i

/ ,’

I/./

JA

’

‘ ,,y“””“--*

0.15 9’ 0

I

I

1 t

I

2

(mid

0.2

FIG. 5. The effect of nigericin on mitochondrial swelling in KCl. L.S. kinetics exhibited by M2+-depleted mitochondria in KC1

medium are shown. The mitochondria (0.1 mg/ml) were added to the assay medium which contained A23187 (10 nmol/mg). Valinomycin (ual, 1nmol/mg) was added at zero time (traces a and c) or at 1 min (traces b and d). Nigericin (nig,0.5 nmol/mg) was added at zero time in trace a and at 0.9 min in trace d. The rates of anion transport calculated from traces a-d are 230,375,425, and 370 nmol of Cl-/ min. mg, respectively. The assay medium is described under “Experimental Procedures.”

0 0

4

8

(JA -JA &Val

FIG.6. Determination of JA,max for C1- uniport. The rate of (trace a) than when it is added at 1 min(trace b ) . Note, C1- transport (Ja,pmol/min. mg) is plotted uersus (JA - JA,o)/[valhowever, that this differencealso appearstorepresent a inomycin] (pmol/nmol. min)where Ja.0 (equal to0.02 pmol/min .mg) change in anion uniport activity associated with K+/H+ ex- is the rate observed in the absence of valinomycin. Analysis of the change, since addition of nigericin stimulates the rate swelling data yields a JA,mal of 434 nmol Cl-/min. mg and a KO, for valinomycin when valinomycin is added a t zero time (trace c) but has no of 0.049 nmol/mg. Mitochondria (0.1 mg/ml) were added to the KC1 effect when valinomycin is added at 1 min (trace d). Thus,a assay medium (described under “Experimental Procedures”) containchange in anion uniport activity is observed in both NH,C1 ing nigericin (1 nmol/mg) and A23187 (10 nmol/mg). Valinomycin was added at 0.2 min. The net salt transport was followed using the and KCl, but the change occurs in opposite directions. L.S. technique, andthe rate of C1- transport was calculated from the Determination of Maximal Anion Fluxes-Having estab- L.S. kinetics as described under “Experimental Procedures.” lished conditions under which reasonably reproducible rates of swelling could be determined, we wished to examine the cating that all these anions can be transported electrophoretselectively of the anion uniporter by determining the maximal ically. The only anions transported at a very rapid rate in fluxes for a variety of different anions. For the purpose of M2+-containing mitochondria are SCN- andClO;, which are quantitative analysis,we have adopteda nonequilibrium ther- generally regarded as lipid soluble (26, 27). The very fast rate modynamic model t o describe the coupling betweenthe fluxes of SCN- transport is attributed to its ability to form lipidof theanionand cation. This model is described inthe soluble ion pairs with the K+-valinomycin complex (28, 29). “Appendix” and predicts thatfor K’ salts there should be an Consistent with this we find that when valinomycin is rehyperbolic relationship between the rateof transport and the placed by nigericin and CCCP, SCN- and C10; are transconcentration of valinomycin if transport of the anion and ported at equal rates (notshown). NO; is frequently thought K’ are electrically coupled. This predictionwas tested by the of as a lipid-soluble anion (8, 10, 30-33); however, as shown experiment shown inFig. 6, in which we have plotted the rate by the data in the table and elsewhere (34), NO; transport in of chloride transport ( J A ) versus ( J A - JA,o)/[valinomycin], normalM2+-containing liver mitochondria is much slower where J A , o is the rate of chloride transport in the absence of than the transport of SCN- and C10;. Swelling observed in valinomycin (see “Appendix”). As predicted by the model, a M2+-depletedmitochondria in the absence of valinomycin (J2) linear relationship is observed. The intercept on the ordinate can be explainedby active K+ orOH- uniport pathways, and yields a J A , m a x for C1- transport of 434 nmol Cl-/min. mg, and values for the maximum fluxes through these pathways (J3) the slope yields a for valinomycin of 0.05 nmol/mg. From can be calculated from J1and J2as described in the “Appenthese data, on the basis of our model, it can be calculated thatdix.” 1 nmol of valinomycin/mg should permit rates of anion transpH Dependence of Anion Uniport-The data of Figs. 3-5 port which are at least 94% of the JA,,,,.= for anions transported strongly suggest that anion uniport activity is influenced by at rates equal to or slower than chloride. Thus, for our survey net exchange of matrix K+ for NH: in NH: salts and the (Table I),we determined the rateof transport induced by this redistribution of K+ via K+/H+ antiport in K+ salts. Two single dose of valinomycin rather than carrying out a full factors contributing to these effects could be: 1)regulation of titration for each anion. anion uniportby matrix pH and 2) achange inAp for chloride. Table I shows transport rates for a wide variety of anions Although it is difficult to demonstrate direct regulation by measured at pH 7.4 under three conditions; Jois the rate in matrix pH, in K+ salts nigericin can be used to equilibrate K+/H+ antiport, thereby ensuring that the matrix pH will the presence of valinomycin alone, J1istherateinthe presence of both valinomycin and A23817, and J2is the rate vary as the medium pH is varied. Furthermore, since the in the presenceof A23187 alone. For all anions tested,except concentration of K’ on both sides of the membrane is relaa minimum. Fig. 7A EDTA, EGTA, TES, gluconate, and glucuronate, M2+ deple- tively high, the pH gradient should be at tion substantially stimulates the rate of anion transport in- contains data showing that the rateof chloride and malonate transport in M2+-depletedmitochondria isstrongly dependent duced by valinomycin (J1/Jo >> 1). Furthermore, formost anion rate transport anions valinomycin substantially stimulates the rate of trans- on pH. Note that at all pHs studied ofthe >> 1) indi- is much lower in normal Mg2+-containing mitochondria. In port induced by divalent cation depletion (Jl/J2

15089

Anion Uniport i n Mitochondria TABLEI Effect of divalent cation depletionon anion transport in mitochondria Jo,Jl, and J2 are rates of anion transport (nmol/min.mg) determined under various conditions. Jo is the rate in normal mitochondria in the presence of valinomycin; J1 is the rate in the presence of both valinomycin and A23817; and J2is the rate in the presence of A23187 alone. J3is the maximum rate of electrophoreticOH- uniport or the maximum rate of endogenous K+ uniport calculated from Jl and J2according to Equation A16 of the “Appendix.” These measurements weremadebyfollowing L.S. kinetics of mitochondria (0.1 mg/ml) added to assay medium containing nigericin (0.5 nmol/mg). When present A23187 (10 nmol/mg) was added at zero time and valinomycin (Val,1 nmol/mg) was addedat 0.5 min. The method used forcalculatingthe transport rates from the L.S. kinetics and the composition of the assay medium are described under “Experimental Procedures.” For the measurementsof HCOi transport acetazolamide (40 PM) was addedto the medium to block carbonicanhydrase. For measurements of Pi transport the mitochondria were pretreated with N-ethylmaleimide (40 nmol/mg) to block the electroneutral Pi--H+ symporter. For measurements of ferrocyanide transport 0.5 mM CN- was added to prevent oxidationof ferrocyanide, and 0.5 mM ascorbate was added to reduce any ferricyanide formed. Anion

SCNNOi

c10; HCO; c1so:-

pi’“Benzenetricarboxylate3Fe(CN)iButy1malonate’Fe(CN):Citrate3Malonate2Malate2Fumarate2p-AminohippurateAdipate2GluconateGlucuronateEDTA3EGTA2TES

Jo

J,

(+Val)

(+Val)

(+A23187)

1017 48 366 16 8.6 11 26 2.6 5.6 16.6 3.2 2.0 4.0 2.6 2.4 4.3 1.5

1618 808 789 630 418 387 267 262 259 212 178 164 140 134 70 26 12