Protein Structural Modeling and Transport Thermodynamics Reveal That Plant Cation–Chloride Cotransporters Mediate Potassium–Chloride Symport

Plant cation–chloride cotransporters (CCCs) are proposed to be Na⁺-K⁺-2Cl⁻ transporting membrane proteins, although evolutionarily, they associate more closely with K⁺-Cl⁻ cotransporters (KCCs). Here, we investigated grapevine (Vitis vinifera L.) VvCCC using 3D protein modeling, bioinformatics, and electrophysiology with a heterologously expressed protein. The 3D protein modeling revealed that the signatures of ion binding sites in plant CCCs resembled those of animal KCCs, which was supported by phylogenomic analyses and ancestral sequence reconstruction. The conserved features of plant CCCs and animal KCCs included predicted K⁺ and Cl⁻-binding sites and the absence of a Na⁺-binding site. Measurements with VvCCC-injected Xenopus laevis oocytes with VvCCC localizing to plasma membranes indicated that the oocytes had depleted intracellular Cl⁻ and net ⁸⁶Rb fluxes, which agreed with thermodynamic predictions for KCC cotransport. The ⁸⁶Rb uptake by VvCCC-injected oocytes was Cl⁻-dependent, did not require external Na⁺, and was partially inhibited by the non-specific CCC-blocker bumetanide, implying that these properties are typical of KCC transporters. A loop diuretic-insensitive Na⁺ conductance in VvCCC-injected oocytes may account for earlier observations of Na⁺ uptake by plant CCC proteins expressed in oocytes. Our data suggest plant CCC membrane proteins are likely to function as K⁺-Cl⁻ cotransporters, which opens the avenues to define their biophysical properties and roles in plant physiology.