Martínez-Zaguilán R, Parnami J, Martinez G M
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Cell Physiol Biochem. 1998;8(3):158-74. doi: 10.1159/000016279.
Based on studies using high-affinity Ca2+ probes (dissociation constant (Kd) = 0.15-0.3 microM), steady-state [Ca2+]in is believed to be in the nanomolar range in most cells. However, probes with lower affinity indicate that [Ca2+]in may increase to micromolar levels during activation of specific cell functions, e.g., contraction. These conclusions rely on accurate knowledge of the Kd of the dyes for Ca2+. Mag-Fura-2 (also known as Furaptra) is a low-affinity Ca2+ indicator (Kd ca. 50 microM) which has been used for such studies. In the present work, Mag-Fura-2 is shown to respond to changes in cytosolic Ca2+ in the submicromolar range. In vitro, and in situ titration of Mag-Fura-2 in A7r5 cells, demonstrate that Mag-Fura-2 exhibits both high- and low-affinity for Ca2+. Moreover, pH affects both high and low affinity Ca2+ binding site. Since Mag-Fura-2 has been used to study Ca2+ within specific subcellular compartments, the present observations indicate that knowledge of factors such as ambient pH of these compartments is required to accurately interpret Ca2+ responses. Furthermore, the sensitivity of Mag-Fura-2 at submicromolar levels must be considered for accurate determination of Ca2+ in specific compartments believed to exhibit high micromolar levels of Ca2+.
基于使用高亲和力钙离子探针（解离常数（Kd）=0.15 - 0.3微摩尔）的研究，大多数细胞内的稳态钙离子浓度被认为处于纳摩尔范围。然而，低亲和力的探针表明，在特定细胞功能激活过程中，如收缩时，细胞内钙离子浓度可能会增加到微摩尔水平。这些结论依赖于对染料与钙离子解离常数的准确了解。Mag - Fura - 2（也称为Furaptra）是一种低亲和力的钙离子指示剂（Kd约为50微摩尔），已用于此类研究。在本研究中，Mag - Fura - 2被证明对亚微摩尔范围内的胞质钙离子变化有反应。在体外以及对A7r5细胞内的Mag - Fura - 2进行原位滴定，结果表明Mag - Fura - 2对钙离子表现出高亲和力和低亲和力。此外，pH值会影响高亲和力和低亲和力的钙离子结合位点。由于Mag - Fura - 2已被用于研究特定亚细胞区室中的钙离子，目前的观察结果表明，需要了解这些区室的环境pH值等因素，才能准确解释钙离子反应。此外，在准确测定被认为呈现高微摩尔水平钙离子的特定区室中的钙离子时，必须考虑Mag - Fura - 2在亚微摩尔水平的灵敏度。
