Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants

Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K⁺ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 ediates Na⁺-coupled K⁺ transport. Arabidopsis AtHKT1, however, transports only Na⁺ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKTI/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K⁺ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K⁺ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K⁺ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K⁺ channel selectivity filter GYG motif. The importance of such filter glycines for K⁺ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na⁺ transporters in plants and that Na⁺/K⁺ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K⁺ channels.