Evidence against Formation of A23187 Dimers and Oligomers in Solution: Photo-Induced Degradation of Ionophore A23187

Abstract

Ionophore A23187 has been proposed to form Ca2+-conducting channels that arise from dimers and oligomers of the compound (e.g., Balasubramanian, S. V., and Easwaran, K. R. K. (1989)Biochem. Biophys. Res. Commun.158, 891–897). To investigate this possibility, the solution behavior of A23187 in chloroform,n-hexane, ethanol, 80% methanol–water, and palmitoyloleoylphosphatidyl choline (POPC) vesicles was investigated using UV–VIS, circular dichroism (CD), and1H NMR techniques. The concentration dependence of the UV–VIS and CD spectra obtained in freshly prepared chloroform solutions indicates that neutral A23187 (HA) exists as a monomer for ionophore concentrations in the range of 50–1000 μm. The cause of time- and concentration-dependent spectral alterations which gave rise to the dimer/channel hypothesis was also investigated. For solutions of 50–1000 μmA23187 in chloroform,n-hexane, and ethanol stored in the dark, no spectral changes were observed for periods of 2 months. However, solutions in these solvents did show time-dependent spectral changes when exposed to light. In 80% methanol–water or phospholipid vesicles, similar spectral changes were observed, even when the solutions were protected from light. Application of TLC and MS methods indicate that the time-dependent spectral changes reflect degradation of A23187, not dimer or oligomer formation. The degradative processes proceed with half-lives ranging from ∼75 to >400 h, and are influenced by several factors, including solvent, exposure to light, ionophore concentration, pH, and the presence of metal ions, EDTA, dissolved oxygen, and a radical inhibitor. The kinetics of Ca2+transport into Quin-2-loaded POPC vesicles by authentic A23187 give no evidence of a channel mechanism, even following a previous and lengthy coincubation of the ionophore with the vesicles.