Protein modelling and thermodynamics reveal that plant cation-chloride cotransporters mediate potassium-chloride symport

Plant cation-chloride-cotransporters (CCC) are proposed to be Na+-K+-2Cl- transporting proteins, although phylogenetically they are closer to K+-Cl- cotransporters (KCC). Conserved features of plant CCC and animal KCC include the presence of predicted K+ and Cl- binding sites, and the absence of a Na+ binding site. Here, we investigated grapevine (Vitis vinifera L.) VvCCC using protein structural modelling and heterologous expression. Our modelling data predicted that 3D folds of VvCCC were more similar to DrNKCC1, but sequences of ion binding sites resembled those of hKCC1. The measurements with VvCCC-injected Xenopus laevis oocytes with VvCCC, localising to plasma membranes, indicated that oocytes were depleted of intracellular Cl-, net 86Rb fluxes, which agreed with thermodynamic predictions for KCC co-transport. 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 - 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 in oocytes. Our data suggest plant CCC proteins are likely to function as K+-Cl- cotransporters, which opens avenues to define their mechanistic and biophysical properties and roles in physiology. HighlightBased on predictive structural and experimental data, this study presents novel findings to reveal that plant cation-chloride-cotransporters are likely to function as K+-Cl- symporters.