Mechanism of Action of “Ruthenium Red” Compounds on Ca2+ Ionophore from Sarcoplasmic Reticulum (Ca2+ + Mg2+)-Adenosine Triphosphatase and Lipid Bilayer*

Sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase was previously shown to have Ca2+-dependent and -selective ionophoric activity when tested in oxidized cholesterol lipid bilayer membranes (Shamoo, A. E., and MacLennan, D. H. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3522). ruthenium red, a known inhibitor of (Ca2+ + Mg2+)-ATPase, is found to inhibit the Ca2+-ionophoric activity associated with (Ca2+ + Mg2+)-ATPase. Furthermore, ruthenium red alone acts as an anion-selective ionophore in lipid bilayers with the the following selectivity sequence for anions: l- greater than Cl-, Br- greater than F- greater than NO3-. The PCl-/PNa+ ratio was approximately 4/l. The presence of ruthenium red in excess of Ca2+ ionophore in lipid bilayer experiments converts the cation selectivity of the bilayer due to Ca2+ ionophore into anion selectivity.

The P,.,-/P,,+ ratio was -4/l. The presence of ruthenium red in excess of Ca"+ ionophore in lipid bilayer experiments converts the cation selectivity of the bilayer due to Ca"+ ionophore into anion selectivity.
Ruthenium red is a powerful inhibitor of Ca2+ transport in mitochondria (l-5). It is also a specific inhibitor of (Ca'+ f Mg'+)-ATPase of red blood cells (S), and of Ca"+ binding and (Ca"' + Mg'+l-ATPase of sarcoplasmic reticulum of rabbit skeletal muscle (7-8). Shamoo and MacLennan (9) have shown that a Caz+-dependent and -selective ionophore is associated with the sarcoplasmic reticulum (Ca'+ + Mg'+)-ATPase from rabbit skeletal muscle. In view of the known action of ruthenium red on (Ca'* + Mg"+)-ATPase we tested its effect on the Gas+ ionophore from (Ca2+ + Mg"i-ATPase. We found that ruthenium red (as an impure compound) has two different effects: (a) it acts as a highly selective anion ionophore in lipid biiayer and converts a cation-selective bilayer (due to the presence of Ca*+ ionophore) into an anion-selective bilayer; (b) it inhibits the Ca"+ ionophore action on bilayers when the Ca"+ ionophore is pretreated with ruthenium red.

MATERIALS AND METHODS
Sarcoplasmic reticulum (Ca" + Mg'+)-ATPase from rabbit skeletal muscle was isolated as described (IO). Succinylation of the whole enzyme greatly increased its aqueous solubility, although it has been shown that the Ca'+-dependent and -selective ionophoric activity is not a result of succinylation (9) However, even if the conductance was increasing during the sweep, no change in the intercept was observed. Thus, the selectivity ratio was independent of conductance once the conductance was higher than the leakage conductance. Before the addition of ruthenium red the Pc;,=JPc, was =4/l (see Curve A in Fig. 2). After the addition of ruthenium red (10m4 M) either the conductance continued to increase (not shown) or decreased slightly and then increased (as shown in  Fig. 2). Thus, the lipid bilayer changes its properties from cationic selectivity to anionic selectivity. Curve C in Fig. 2 indicates that ruthenium red endows the lipid bilayer with anion selectivity in the absence of (Ca2+ + Mg2+)-ATPase ionophore similar to that in the presence of the Ca2+ ionophore and ruthenium red. Next, we wanted to differentiate between the following modes of ruthenium red action: (a) ruthenium red might overwhelm the Ca2+ ionophore in the bilayer with anion-selective pathways, (b) ruthenium red might convert the cation-selective channel within the ionophore into an anion-selective channel, and (c) ruthenium red might inhibit the Ca2+ ionophore and also act as an anion ionophore.
We then proceeded to preincubate concentrated Ca2+ ionophore (2.6 mg/ml) with the same concentration of ruthenium red as in Fig. 1 (lo-* M). Aliquots of the pretreated Ca2+ ionophore were then added to the bathing fluid of the bilayer to give 2.6 x 10e3 mg/ml of Ca*+ ionophore. The final concentration of ruthenium red (lo-' M) in the bathing fluid due to the addition of the pretreated Ca'+ ionophore was less than (VIOOO) that in Fig. 1. We have determined that at such low concentration ruthenium red has no direct effect on bilayer conductance. The result of this experiment was that there was no increase in lipid bilayer conductance.
This indicates that ruthenium red completely blocks the action of Ca2+ ionophore.
We increased the pretreated Ca2+ ionophore concentration to a level 5 times higher than that normally used in order to look for a strong effect on lipid bilayer conductance. The results even at such high dosage were negative. The aforementioned results indicate that ruthenium red has two different effects: (a) it inhibits the Ca2+ ionophore and (b) it acts as an anion-selective ionophore in lipid bilayer.
In order to measure the diffusion potential we started with equal concentrations of ions on both sides of the membrane followed by the addition of small volumes of highly concentrated salt to one side to create a salt gradient. It was observed that, after reaching a steady state conductance, a further increase in conductance occurred following the formation of the salt gradient (Fig. 3). In A both sides of the bilayer bathing fluid contained 2.6 X lo-$ mg/ml of (Ca*+ + MgP+l-ATPase i onophore i n addition to Ca*+. In S, the conditions were as i n A plus lo-' M ruthenium red. In C, lo-' M ruthenium red was present, but there was no (Ca*+ + MgZ+)-ATPase i onophore. The experimental measurements were made i n a manner similar to that described for Fig. 1. chloride to either Ca"+ or Na'+ when measured by the diffusion potential. The lower selectivity value for PC,-/PNa+ than for P&PC,*+ may be due to leakage of Na+. To find the anionselectivity ratio of the bilayer in the presence of ruthenium red we tested the bi-ionic potential in the presence of 5 mM CaCl, + 5 mM histidine (pH 7.3) versus 5 mM Ca(NO& (or CaBr, or CaI,) + 5 mM histidine (pH 7.3). In the case of fluoride we used 5 mM NaCl versus 5 mM NaF (Table II).
In the case of Ca(NO,), versus CaCl, we found that the bilayer had to be formed from the CaCl, side in order to achieve an increase in conductance when ruthenium red was present on both sides of the membrane. The rate of increase in bilayer conductance due to ruthenium red under such conditions was lower than that when CaCl, and ruthenium red were present on both sides of the bilayer. As a matter of fact, if Ca(N0J2, CaCl*, and ruthenium red were present on both sides of the bilayer, no increase in conductance was observed. The presence of compounds like sulfate, nitrate, and oxalate on one or both sides of the bilayer at concentrations equal to CaCl, caused the inhibition of ruthenium red action on the bilayer when ruthenium red was present on both sides of the bilayer. ATP was also found to be inhibitory to the effect of ruthenium red on the bilayer. This may indicate that multivalent anions form complexes with ruthenium red (6+) preventing its incorporation into the bilayer or its complexation with monovalent anions like chloride. The ruthenium red in a multivalent anion complex may become immobile in the membrane. This may explain some of the inconsistent results ob- The experimental measurements were made i n a manner similar to that described for Fig. 1.

Tas~x I
Catbn:antin selsctiuity Cation:anion selectivity ratio of oxidized cholesterol black l i pi d membrane i n the presence of lo-' ruthenium red + 5 mM histidine (pH 7.3) on both sides of the bilayer and the appropriate ions. In the first row we had 50 rnM CaCl, i nsi de versus 5 rnM CaCI, outside.
In the second row we had 50 mr.r NaCl uersas 5 rnM NaCI. The experimental measurements were made i n a manner similar to that described for Fig. 1