Fudo Satoshi, Verkhovskaya Marina, Di Scala Coralie, Rivera Claudio, Kajander Tommi
Institute of Biotechnology, HiLIFE, University of Helsinki, Finland.
Neuroscience Center, HiLIFE, University of Helsinki, Finland.
FEBS Open Bio. 2025 Sep;15(9):1532-1545. doi: 10.1002/2211-5463.70063. Epub 2025 Jun 13.
The cation-chloride cotransporter (CCC) family includes ion symporters that cotransport monovalent cations and Cl, playing a crucial role in controlling cytoplasmic ion content. K-Cl cotransporters (KCCs) facilitate the symport of ions across the plasma membrane. The CCCs participate in various physiological processes, such as transepithelial ion transport and regulation of cell volume. Among KCCs, KCC2 has unique and essential functions in the central nervous system. KCC from Drosophila melanogaster (DmKCC) is an ortholog of mammalian KCCs. Its critical role in neuronal transmission has been demonstrated. Also, the cnidarian Hydra vulgaris has a functional KCC (HvKCC). Comparative analyses of these transporters with vertebrate counterparts can provide insights into the mechanism of KCC ion transport, regulation, and evolution. Thus, here we purified DmKCC and HvKCC and characterized their biophysical properties using differential scanning fluorimetry and light scattering. We evaluated their functionality in cells and developed a method to study ion transport with flame photometry. Further, a fluorescence-based assay for DmKCC reconstituted into proteoliposomes was developed. The activity of DmKCC was found to be dependent on Ca, which is reminiscent of some other chloride transport protein families and potentially important for the KCC protein family overall.
阳离子-氯离子共转运体（CCC）家族包括共转运单价阳离子和氯离子的离子同向转运体，在控制细胞质离子含量方面发挥着关键作用。钾-氯共转运体（KCCs）促进离子跨质膜的同向转运。CCC参与多种生理过程，如跨上皮离子转运和细胞体积调节。在KCC中，KCC2在中枢神经系统中具有独特且重要的功能。果蝇（Drosophila melanogaster）的KCC（DmKCC）是哺乳动物KCC的直系同源物。其在神经元传递中的关键作用已得到证实。此外，水螅（Hydra vulgaris）有一种功能性的KCC（HvKCC）。将这些转运体与脊椎动物的对应物进行比较分析，有助于深入了解KCC离子转运、调节和进化的机制。因此，我们在此纯化了DmKCC和HvKCC，并使用差示扫描荧光法和光散射对其生物物理特性进行了表征。我们评估了它们在细胞中的功能，并开发了一种用火焰光度法研究离子转运的方法。此外，还开发了一种基于荧光的测定法，用于检测重组到蛋白脂质体中的DmKCC。结果发现DmKCC的活性依赖于钙，这与其他一些氯化物转运蛋白家族的情况相似，可能对整个KCC蛋白家族具有重要意义。
