Characterization of ligand binding to mitochondrial benzodiazepine receptors

JD Hirsch, CF Beyer, L Malkowitz, CC Loullis and AJ Blume

Department of Central Nervous System Research, American Cyanamid Company, Pearl River, New York 10965.

We have evaluated the affinity and density of binding sites for [3H]Ro5- 4864 and [3H]PK11195 in intact and fragmented rat kidney mitochondria. These sites are known as peripheral-type or mitochondrial benzodiazepine receptors (MBR) and the preceding paper provided evidence that they function in vitro as modulators of mitochondrial respiratory control (1). In this report, MBR density, localization, and ligand specificity were investigated. In intact mitochondria, there were approximately the same number of binding sites for [3H]PK11195 as for [3H]Ro5-4864, and their apparent Kd values were identical. However, in mitochondrial fragments, there were 80% more binding sites for [3H]Ro5-4864 than for [3H]PK01195. Rat kidney mitochondria were fractionated by decompression and digitonin-based methods into outer and inner membrane-containing fractions before and after incorporation of the MBR-specific photoaffinity ligand [3H]PK14105. Assays of selective mitochondrial membrane markers and [3H]Ro5-4864 binding or specifically bound [3H]PK14105 revealed that the receptors were found in the mitochondrial outer membrane. We also evaluated the binding of a large number of structurally and pharmacologically diverse compounds to MBR by studying their ability to inhibit the binding of both 3H- ligands. These compounds had affinities ranging from 0.015 to 100 microM and, with a few exceptions, were similar in their abilities to bind to MBR in intact and fragmented mitochondria. However, there was considerable variation in the ratios between drug potencies at displacing [3H]Ro5-4864 and [3H]PK11195. This represents a new form of evidence that these two radioligands do not label identical sites on the receptor. Thirteen of the drugs, including [3H]Ro5-4864 and [3H]PK11195, were analyzed as to the nature of the inhibition and, with only two exceptions, were competitive inhibitors. One drug, Konig's polyanion, was uncompetitive whereas the other, cyclosporin A, was a noncompetitive inhibitor. These studies revealed several new classes of MBR ligands and suggest that the relationship between ligand structure and binding affinity is highly complex.

Volume 35, Issue 1, pp. 164-172, 01/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				H. Yamada, Y. Matsuki, T. Yamaguchi, and K. Oguri Effect of a Ligand Selective for Peripheral Benzodiazepine Receptors on the Expression of Rat Hepatic P-450 Cytochromes: Assessment of the Effect In Vivo and in a Hepatocyte Culture System Drug Metab. Dispos., November 1, 1999; 27(11): 1242 - 1247. [Abstract] [Full Text]

				J. D. Waterfield, E. G. McGeer, and P. L. McGeer The peripheral benzodiazepine receptor ligand PK 11195 inhibits arthritis in the MRL-lpr mouse model Rheumatology, November 1, 1999; 38(11): 1068 - 1073. [Abstract] [Full Text] [PDF]

				M. E. Whalin and K. E. Krueger Identification and Purification of a 10-Kilodalton Protein Associated with Mitochondrial Benzodiazepine Receptors J. Biol. Chem., September 1, 1995; 270(35): 20285 - 20291. [Abstract] [Full Text] [PDF]