The kinetic properties of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from canine cerebellum microsomes were investigated by stopped-flow (rapid mixing) techniques. At room temperature, saturating concentrations of InsP3 released Ca2+ with a half-time of 200 msec, the initial release rate being about 34 nmoles Ca2+/sec/mg of protein, and the first-order rate constant about 8 sec-1. Thus, rates of Ca2+ release in vitro are kinetically compatible with in vivo intracellular Ca2+ transients. The apparent Km for InsP3-induced Ca2+ release was 0.9 æM and the Hill coefficient (n) was 1.2. Under identical experimental conditions, specific [3H]InsP3 binding was measured, and Bmax of 3.5 pmol InsP3 bound/mg of protein, Kd of 52 nM and n of 1.1 were determined. Thus, both rapid InsP3-induced Ca2+ release and [3H]InsP3 binding to a single class of independent receptor sites appeared not to be cooperative. Discrepancies between Km for release and Kd for InsP3 binding are reconciled by a model for the InsP3-gated Ca2+ release channel in which at least four InsP3 binding sites must be occupied before the channel can open. This model is consistent with the tetrameric subunit structure of the channel (Maeda et al., EMBO J. 9, 61-67, 1990; J. Biol. Chem. 266, 1109-1116, 1991).
Kinetic model of quantal calcium release from intracellular calcium stores
VOLPE, POMPEO
1998
Abstract
The kinetic properties of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from canine cerebellum microsomes were investigated by stopped-flow (rapid mixing) techniques. At room temperature, saturating concentrations of InsP3 released Ca2+ with a half-time of 200 msec, the initial release rate being about 34 nmoles Ca2+/sec/mg of protein, and the first-order rate constant about 8 sec-1. Thus, rates of Ca2+ release in vitro are kinetically compatible with in vivo intracellular Ca2+ transients. The apparent Km for InsP3-induced Ca2+ release was 0.9 æM and the Hill coefficient (n) was 1.2. Under identical experimental conditions, specific [3H]InsP3 binding was measured, and Bmax of 3.5 pmol InsP3 bound/mg of protein, Kd of 52 nM and n of 1.1 were determined. Thus, both rapid InsP3-induced Ca2+ release and [3H]InsP3 binding to a single class of independent receptor sites appeared not to be cooperative. Discrepancies between Km for release and Kd for InsP3 binding are reconciled by a model for the InsP3-gated Ca2+ release channel in which at least four InsP3 binding sites must be occupied before the channel can open. This model is consistent with the tetrameric subunit structure of the channel (Maeda et al., EMBO J. 9, 61-67, 1990; J. Biol. Chem. 266, 1109-1116, 1991).
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