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

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Abstract
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 mediates 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 AtHKT1/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.