Biophysical and functional characterization of K⁺-Cl^-co-transporters fromDrosophila melanogasterandHydra vulgaris

Abstract

The cation-chloride co-transporter (CCC) superfamily includes ion symporters, which co-transport monovalent cations and Cl-. CCCs have crucial roles in shaping signalling and neuronal connectivity in the vertebrate brain. K+-Cl- co-transporters (KCCs) are a subfamily of CCCs and carry out the symport of K+ and Cl− ions across the plasma membrane. The KCC proteins are involved in various physiological processes, such as cell volume regulation, transepithelial ion transport, synapse formation and signal transmission, and blood pressure regulation.Among KCCs, KCC2 has gained attention because of its unique and crucial functions in the central nervous system neuronal network. Loss of activity of this transporter has been associated with several neurological disorders including schizophrenia, epilepsy, and chronic pain.On the other hand, only a limited number of studies of KCCs have been published for invertebrates. Among invertebrate proteins, the Drosophila melanogaster KCC (DmKCC) has been studied most and suggested critical for neuronal transmission. Also Cnidarian Hydra vulgaris has been shown to have a functional KCC (HvKCC). Comparative analyses of these transporters with vertebrate ones and understanding functional and biophysical aspects of them as a model system can help understand the KCC mechanism of ion transport and its regulation and evolution broadly.In this study, we chose DmKCC and HvKCC as model systems and purified DmKCC and HvKCC from Sf9 insect cells and characterized their biophysical properties with differential scanning fluorimetry and light scattering techniques. We tested their functionality using a fluorescence assay and developed a method to measure recombinant KCC ion transport activity with flame photometry.