Hippocalcin (HPCA) is a neuronal calcium sensor (NCS) protein that provides intracellular signaling via its Ca2 + -dependent translocation from the cytosol to the plasma membrane. Although such translocation of HPCA and other NCS proteins is well established, no quantitative estimates of HPCA distribution between the cytosol and plasma membrane at the basal level of the intracellular free calcium concentration ([Ca 2+ ] i ) have been obtained. Thus, it is still unknown whether HPCA regulates its plasma membrane targets under resting conditions. In this work, we have evaluated this distribution in living cells at the basal level of [Ca 2+ ] i , comparing HPCA spatial distribution with that of exogenously expressed membrane protein, EYFP-Mem. Using fluorescence recovery after photobleaching, we showed that the diffusion coefficient of fluorescently tagged HPCA in the dendrites of hippocampal neurons (~40 μm 2 /sec) is within the range of values typical of cytosolic proteins. The coefficient was about 10-fold higher than that for EYFP-Mem. Both results indicate that HPCA is mainly localized in the cytosol of the dendrites of hippocampal neurons. Next, direct calculations for HPCA fractions in the cytosol and plasma membrane at the basal level of [Ca 2+ ] i demonstrated that the plasma membrane fraction of HPCA does not exceed 8%. Altogether, our results suggest that although being mainly cytosolic, HPCA may potentially signal its plasma membrane targets under resting conditions. New approaches used for obtaining such estimates can be applied for quantitative evaluation of NCS protein distribution in living cells under different experimental conditions and for the development of precise biophysical models of their signaling.

中文翻译：

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活细胞的细胞质和质膜之间的海马钙素分布

Hippocalcin (HPCA) 是一种神经元钙传感器 (NCS) 蛋白，通过其 Ca2 + 依赖性从细胞溶质到质膜的易位提供细胞内信号。尽管 HPCA 和其他 NCS 蛋白的这种易位已被很好地确定，但尚未获得在细胞内游离钙浓度 ([Ca 2+ ] i ) 的基础水平上细胞质和质膜之间 HPCA 分布的定量估计。因此，HPCA 是否在静息条件下调节其质膜靶点仍是未知数。在这项工作中，我们在 [Ca 2+ ] i 的基础水平评估了活细胞中的这种分布，将 HPCA 空间分布与外源表达的膜蛋白 EYFP-Mem 的空间分布进行了比较。利用光漂白后的荧光恢复，我们表明，海马神经元树突中荧光标记的 HPCA 的扩散系数 (~40 μm 2 /sec) 在细胞质蛋白的典型值范围内。该系数比 EYFP-Mem 的系数高约 10 倍。这两个结果都表明 HPCA 主要位于海马神经元树突的细胞质中。接下来，在 [Ca 2+ ] i 的基础水平直接计算细胞质和质膜中的 HPCA 分数表明 HPCA 的质膜分数不超过 8%。总而言之，我们的结果表明，虽然主要是细胞溶质，但 HPCA 可能会在静息条件下向其质膜目标发出信号。