Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes.
The binding of [3H]inositol hexakisphosphate ([3H] InsP6) to rat cerebellar membranes has been characterized with the objective of establishing the role, if any, of a membrane protein receptor. In the presence of EDTA, we have previously identified an InsP6-binding site with a capacity of approximately 20 pmol/mg protein (Hawkins, P. T., Reynolds, D. J. M., Poyner, D. R., and Hanley, M. R. (1990) Biochem. Biophys. Res. Commun. 167, 819-827). However, in the presence of 1 mM Mg2+, the capacity of [3H]InsP6 binding to membranes was increased approximately 9-fold. This enhancing effect of Mg2+ was reversed by addition of 10 microM of several cation chelators, suggesting that the increased binding required trace quantities of other metal cations. This is supported by experiments where it was possible to saturate binding by addition of excess membranes, despite not significantly depleting radioligand, pointing to removal of some other factor. Removal of endogenous cations from the binding assay by pretreatment with chelex resin also prevents the Mg(2+)-induced potentiation. Consideration of the specificity of the chelators able to abolish this potentiation suggested involvement of Fe3+ or Al3+. Both these ions (but not several others) were able to increase [3H]InsP6 binding to chelex-pretreated membranes at concentrations of 1 microM. It is possible to demonstrate synergy between Fe3+ and Mg2+ under these conditions. We propose that [3H]InsP6 may interact with membranes through non-protein recognition, possibly via phospholipids, in a manner dependent upon trace metals. The implications of this for InsP6 biology are considered.
Poyner
D R
DR
Medical Research Council Centre, Cambridge, United Kingdom.
Cooke
F
F
Hanley
M R
MR
Reynolds
D J
DJ
Hawkins
P T
PT
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D004298:Dopamine / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D006719:Homovanillic Acid / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009638:Norepinephrine / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010833:Phytic Acid / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D014316:Tritium; D014443:Tyrosine / Q000494:pharmacology
D007295:Inositol Phosphates; D014316:Tritium; D010656:Phenylephrine; D014443:Tyrosine; D004533:Egtazic Acid; D010833:Phytic Acid; D011433:Propranolol; D008274:Magnesium; D007545:Isoproterenol; D002118:Calcium; D004298:Dopamine; D009638:Norepinephrine; D006719:Homovanillic Acid; D004837:Epinephrine