Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+

Abstract

We have characterized in detail the Ca# + -dependent inhibition of [$H]Ins(1,4,5)P $ ([$H]InsP $ ) binding to sheep cerebellar microsomes, over a short duration (3 s), with the use of a perfusion protocol. This procedure prevented artifacts previously identified in studies of this Ca# + effect. In a cytosol-like medium at pH 7.1 and 20 mC, a maximal inhibition of approx. 50 % was measured. Both inhibition and its reversal were complete within 3 s. Ca# + decreased the affinity of the receptor for InsP $ by approx. 50 % (K d 146p24 nM at pCa 9 and 321p56 nM at pCa 5.3), without changing the total number of binding sites. Conversely, increasing the [$H]InsP $ concentration from 30 to 400 nM tripled the IC &! for Ca# + and decreased the maximal inhibition by 63 %. This is similar to a partial competitive inhibition between InsP $ binding and inhibitory Ca# + binding and is consistent with InsP $ and

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

“…We show here that K + , the predominant inorganic cation in cells, has a strong effect on the binding of InsP 3 to sheep cerebellar microsomes, a subcellular fraction containing mostly type 1 InsP 3 R [7,38]. Increasing the concentration of K + or other inorganic cations in the medium surrounding the membranes increased both the K d and B max for InsP 3 binding.…”

“…Conversely, the removal of K + causes a rapid reversal of the increase in B max , suggesting that the high-affinity sites are reconverted into low-affinity undetectable sites. Thus InsP 3 R is present in cerebellar membranes in at least two interconvertible states with different affinities for InsP 3 (independently of the affinity regulators, Ca 2+ [7] and InsP 3 [35]). K + favours a state with nanomolar affinity, whereas, in the absence of this cation, InsP 3 R is mainly in a conformation with a lower affinity (with a K d presumably > 1 µM).…”

“…InsP 3 binding to cerebellar microsomes was measured at 20 • C by means of a perfusion protocol similar to that described elsewhere [7,[33][34][35]. Microsomal membranes were thawed and diluted in an ice-cold medium (buffer A: 25 mM Hepes/Tris, pH 7.2, 1 mM H 3 PO 4 , 0.3 mM EDTA, 0.3 mM EGTA, 2 µg/ml leupeptin and 0.2 mM PMSF).…”

“…InsP 3 and Ca 2+ are the major determinants of the channel activity of the three InsP 3 R isoforms. These two regulators may stimulate or inhibit InsP 3 R activity [2][3][4][5][6][7]. Control by these agents is thought to be essential for the creation of complex Ca 2+ signals with various patterns [1].…”

“…A recent study showed that, in the functional InsP 3 R, individual binding sites may be located very close together, within 2 nm of each other [20]. Cytosolic Ca 2+ controls channel activity in a biphasic manner [3,21] and inhibits InsP 3 binding to InsP 3 R 1 [3,7,22,23].…”