Mezna M, Michelangeli F
School of Biochemistry, University of Birmingham, Edgbaston, United Kingdom.
J Biol Chem. 1995 Nov 24;270(47):28097-102. doi: 10.1074/jbc.270.47.28097.
The effects of the alkali metal ions Na+, K+, Rb+, and Cs+ on ATP-dependent Ca2+ uptake, [3H]Inositol 1,4,5-trisphosphate (InsP3) binding, and quantal InsP3-induced Ca2+ release were investigated using rat cerebellar microsomes. Both the ion species and concentration affected the ability of the microsomes to support Ca2+ uptake with K+ being mot effective (3.8 nmol of Ca2+/min/mg at 100 mM K+). The order of efficacy of the other ions was as follows: K+ > Na+ > Rb+ = Cs+ >> Li+. The binding of [3H]InsP3 to cerebellar microsomes was, however, affected little by the presence of these ions. All these alkali metal ions (except Li+) supported InsP3-induced Ca2+ release at concentrations above 25 mM; however, the extent of Ca2+ release (expressed as a percent Ca2+ release compared with that released by the ionophore A23187) was dependent upon the ion species present. Again K+ was more potent than the other ions at facilitating InsP3-induced Ca2+ release (order of efficacy: K+ > Rb+ > Na+ > Cs+), although the concentration of InsP3 required to induce half-maximal Ca2+ release (IC50) was not significantly altered. Over the ion concentration range tested (25-100 mM), the extent of InsP3-induced Ca2+ release with both K+ and Rb+ increased in a linear fashion, while Na+ showed only a slight increase and Cs+ showed no increase over this range. The effect of K+ concentration on quantal Ca2+ release was to alter the extent of release rather than the IC50 InsP3 concentration. Using stopped-flow techniques, the effects of InsP3 and K+ concentrations on the kinetics of InsP3-induced Ca2+ release were shown to exhibit a monoexponential process in this microsomal preparation. The rate constants for Ca2+ release increased with InsP3 concentration (0.11 s-1 at 0.02 microM InsP3 to 0.5 s-1 at 40 microM InsP3); however, the relationship between the fractional extent of release and rate constants for release did not change in a similar way with InsP3 concentration. Although the fractional extent of Ca2+ release increased with K+ concentration, the rate constants for release over this K+ concentration range were unaffected. This observation leads us to question the role of K+ as a counter ion required for Ca2+ release, and we therefore postulate a role for K+ (and the other alkali metal ions) as a "co-factor" required for channel opening.
利用大鼠小脑微粒体研究了碱金属离子Na⁺、K⁺、Rb⁺和Cs⁺对ATP依赖性Ca²⁺摄取、[³H]肌醇1,4,5 - 三磷酸（InsP3）结合以及量子化InsP3诱导的Ca²⁺释放的影响。离子种类和浓度均影响微粒体支持Ca²⁺摄取的能力，其中K⁺最有效（在100 mM K⁺时为3.8 nmol Ca²⁺/分钟/毫克）。其他离子的效力顺序如下：K⁺＞Na⁺＞Rb⁺ = Cs⁺ >> Li⁺。然而，这些离子的存在对[³H]InsP3与小脑微粒体的结合影响很小。所有这些碱金属离子（Li⁺除外）在浓度高于25 mM时均支持InsP3诱导的Ca²⁺释放；然而，Ca²⁺释放的程度（以与离子载体A23187释放的Ca²⁺相比的Ca²⁺释放百分比表示）取决于存在的离子种类。同样，在促进InsP3诱导的Ca²⁺释放方面，K⁺比其他离子更有效（效力顺序：K⁺＞Rb⁺＞Na⁺＞Cs⁺），尽管诱导半数最大Ca²⁺释放（IC50）所需的InsP3浓度没有显著改变。在所测试的离子浓度范围（25 - 100 mM）内，K⁺和Rb⁺诱导的InsP3诱导的Ca²⁺释放程度呈线性增加，而Na⁺在此范围内仅略有增加，Cs⁺则无增加。K⁺浓度对量子化Ca²⁺释放的影响是改变释放程度而非InsP3浓度的IC50。使用停流技术表明，在这种微粒体制剂中，InsP3和K⁺浓度对InsP3诱导的Ca²⁺释放动力学表现出单指数过程。Ca²⁺释放的速率常数随InsP3浓度增加（在0.02 μM InsP3时为0.11 s⁻¹，在40 μM InsP3时为0.5 s⁻¹）；然而，释放程度分数与释放速率常数之间的关系并未随InsP3浓度以类似方式变化。尽管Ca²⁺释放程度分数随K⁺浓度增加，但在此K⁺浓度范围内释放的速率常数不受影响。这一观察结果使我们质疑K⁺作为Ca²⁺释放所需抗衡离子的作用，因此我们推测K⁺（以及其他碱金属离子）作为通道开放所需的“辅助因子”的作用。
