Regulation of the cerebellar inositol 1,4,5-trisphosphate receptor by univalent cations

Abstract

In the present study we investigated the effects of K+ and other univalent cations on [3H]InsP3 {[3H]Ins(1,4,5)P3} binding to sheep cerebellar microsomes. In equilibrium binding experiments performed over 4 s at pH 7.1 and 20 °C, the addition of K+ to the binding medium decreased the affinity and increased the total number of binding sites for InsP3 in a dose-dependent manner. At low InsP3 concentration (0.5 nM) these effects resulted in a biphasic dose–response curve, with maximal binding at about 75 mM K+. In contrast, the dose–response curve calculated for InsP3 at the physiological concentration of 5 μM, was linear up to 200 mM K+. Univalent inorganic cations stimulated [3H]InsP3 binding to various extents, with the following descending order of efficiency at 75 mM: Cs+≈Rb+≈K+>Na+>Li+. The effect of K+ on InsP3R affinity was rapidly reversed upon cation removal. We were therefore also able to demonstrate that K+ increased Bmax (maximal specific binding) by pre-treating microsomes with K+ before measuring [3H]InsP3 binding in the absence of that cation. The increase in Bmax was reversible, but this reversal occurred less rapidly than the change in affinity. These results are consistent with a process by which K+ reversibly converted very low-affinity sites into sites with higher affinity, making them detectable in competitive binding experiments. They suggest that interconversion between these two affinity states constitutes the basis of a K+-controlled regulatory mechanism for cerebellar InsP3R.