Identification of an imidazoline binding protein: Creatine kinase and an imidazoline-2 binding site

Reference:

Kimura, A., Tyacke, R. J., Robinson, J. J., Husbands, S. M., Minchin, M. C. W., Nutt, D. J. and Hudson, A. L., 2009. Identification of an imidazoline binding protein: Creatine kinase and an imidazoline-2 binding site. Brain Research, 1279, pp. 21-28.

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Official URL:

http://dx.doi.org/10.1016/j.brainres.2009.04.044

Abstract

Drugs that bind to imidazoline binding proteins have major physiological actions. To date, three subtypes of such proteins, I-1, I-2 and I-3, have been proposed, although characterisations of these binding proteins are lacking. I-2 binding sites are found throughout the brain, particularly dense in the arcuate nucleus of the hypothalamus. Selective I-2 ligands demonstrate antidepressant-like activity and the identity of the proteins that respond to such ligands remained unknown until now. Here we report the isolation of a similar to 45 kDa imidazoline binding protein from rabbit and rat brain using a high affinity ligand for the I-2 subtype, 2-BFI, to generate an affinity column. Following protein sequencing of the isolated similar to 45 kDa imidazoline binding protein, we identified it to be brain creatine kinase (B-CK). B-CK shows high binding capacity to selective I-2 ligands; [H-3]-2-BFI (5 nM) specifically bound to B-CK (2330 +/- 815 fmol mg protein(-1)). We predicted an I-2 binding pocket near the active site of B-CK using molecular modelling. Furthermore, B-CK activity was inhibited by a selective I-2 irreversible ligand, where 20 mu M BU99006 reduced the enzyme activity by 16%, confirming the interaction between B-CK and the I-2 ligand. In summary, we have identified B-CK to be the similar to 45 kDa imidazoline binding protein and we have demonstrated the existence of an I-2 binding site within this enzyme. The importance of B-CK in regulating neuronal activity and neurotransmitter release may well explain the various actions of I-2 ligands in brain and the alterations in densities of I-2 binding sites in psychiatric disorders.

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