Monovalent cation-A23187 equilibria in MeOHH2O solutions and on phospholipid vesicles

Abstract Although the value of ionophore A23187 as a research tool arises from its transport specificity for divalent cations, the source and limits of this specificity have not been thoroughly studied. Previous work has shown that A23187 can complex monovalent cations [1], extract them to a bulk organic phase [2] and produce monovalent cation transport across certain biological membranes [3]. To elucidate the basis of the normal divalent cation specificity of this ionophore, detailed studies of the mechanism of monovalent cation transport will be necessary. In this communication we report equilibrium constants for complexation reactions involved in the transport of monovalent cations by this compound. Experimental Complexation constants in MeOHH2O mixtures were determined from absorbance measurements whereas fluorescence methods were employed for suspensions of small, unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC). Nonaqueous pH* values in MeOHH2O mixtures were established and measured as described previously [4]. DMPC vesicles were prepared by sonication [5] and purified by ultracentrifugation [6]. Results Table I shows 1:1 complexation constants of the ionophore with Li+ and Na+. The values in MeOHH2O mixtures were determined as conditional constants, K′MA, by titration of A23187 with excess metal ion over the pH* range of 6–10. The equilibrium constants, K*MA, for the reaction M+ + A− ⇄ MA were obtained from the conditional values utilizing the relationship K′MA = K*MA (1 + K*H1a*H), where K*H1 and a*H are the mixed-mode protonation constant and hydrogen ion activity in a given solvent, respectively. The complexation constant in suspensions of DMPC vesicles, KbMA, is defined by the reaction M+aq + A−b ⇄ MAb t001 . Complex Formation Constants of Ionophore A23187 with Li+ and Na+ at 25 °C. a Medium log KMA M = Li+ M = Na+ 65% MeOHH2O b 2.53 ± 0.04 c 1.95 ± 0.08 80% MeOHH2O 3.08 ± 0.05 2.36 ± 0.07 95% MeOHH2O 3.54 ± 0.03 - 100% MeOH d 4.1 3.4 DMPC vesicles 3.22 ± 0.04 - a Ionic strength maintained at 0.05 M with (C2H54NCIO4, buffer composition similar to that cited in Ref. 4. b Weight % MeOH. c Error given as 1 std. dev. d Taken from Ref. 1, μ ∼ 0. where the subscripts aq and b denote solution and membrane-bound species, respectively. Constants for the bound compound were determined under conditions where the fraction of ionophore in the aqueous phase is small. The species A−b was generated by utilizing a high aqueous phase pH and then titrated with excess metal ion. The effect of temperature on KbLiA is shown in Fig. 1 as a Vant'Hoff plot. The pronounced discontinuity in these data is located near the gel to liquid phase transition temperature (Tc) of DMPC vesicles (23 °C). The ΔH and ΔS values obtained from these data above Tc, are equal to −6.4 Kcal/mol and −6.7 cal/degree mol, respectively. Discussion The smooth increase in log K*MA with decreasing solvent polarity is similar to trends observed for other polyether ligands [7] and analogous to the effect of solvent polarity on K*H1 [4]. The stability constants of the LiA complex are 4–5 fold greater than the NaA complex, in contrast to other carboxylic acid ionophores [1]. The stability of the Li+ complex on vesicle membranes is similar to the solution value in 80–85% MeOHH2O, as is the case with the protonation constant [4]. This finding suggests a predominantly interfacial location of the complex. However, the discontinuity on log KbLiA indicates that portions of complex penetrate the membrane acyl group region [8]. The present conditions do not allow observation of the 2:1, ionophore:cation, complexes with monovalent cations which were observed in bulk solvent extraction experiments [2]. These higher order complexes may be of greater for the transport of monovalent cations by A23187 since preliminary experiments show the rate of A23187-dependent Li+ transport into mitochondria is a second order function of the ionophore level.