Abstract
Incubation of cerebellar microsomes with d-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01 1 microM), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nm [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5 2. 5-fold) was obtained with 1 mum InsP3, and the EC50 was 60 +/- 5 nm. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nm [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 microM InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nm, Bmax = 28 pmol/mg of protein; after preincubation with 1 microM InsP3: KD = 53 nm, Bmax = 32 pmol/mg of protein). The two states of the receptor bound InsP3 with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP3 pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent KD = 66 nm). These results report a new regulatory process of the InsP3 receptor in cerebellum occurring independently of Ca2+ and on a relatively long time scale.
Highlights
Channel located in the endoplasmic reticulum (Taylor and Richardson, 1991; Ferris and Snyder, 1992; Berridge, 1993)
Studies of InsP3 binding and InsP3-induced Ca2ϩ release (IICR) with permeabilized cells or diverse receptor preparations including microsomes have allowed the identification of a variety of regulatory mechanisms for the InsP3 receptor
It has been shown that IICR activation in permeabilized hepatocytes is followed by a period of inactivity dependent on the duration of exposure to InsP3 (Hajnoczky and Thomas, 1994)
Summary
Sheep cerebellar microsomes were prepared as described previously (Hannaert-Merah et al, 1994). In the first series of experiments, InsP3 was removed by centrifuging the preincubation mixture at 36,000 ϫ g for 1 h and washing membranes twice with ice-cold MI. The membrane suspension preincubated with or without InsP3 was layered onto a Whatman GF/C glass fiber filter and washed with 10 ml of MI. Four hundred microliters of the mixture were transferred onto a GF/C glass fiber filter which was rinsed with 1 ml of an ice-cold washing medium (25 mM Hepes, 250 mM sucrose, 1 mM NaH2PO4, 1 mM EDTA, pH 7.1). The filter was shortly rinsed with 1 ml of the ice-cold washing medium. The perfusion rate of outflow was adjusted so that the washing lasted less than 0.5 s This procedure removed almost all [3H]InsP3 trapped in the GF/C glass fiber filter.
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