Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple
gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth
transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of
ion transporters require a barrier to prevent free
diffusion of
ions down their
electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (
Synechocystis KtrB-mediated K(+)
uniporter, Arabidopsis AtHKT1-mediated Na(+)
uniporter and
wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in
E. coli,
electrophysiological measurements in
oocytes and growth rates of
E. coli and
yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to
cation transport activity. This positive residue and the conserved Arg both face towards the
ion conducting pore side. We introduced an atomic-scale homology model for predicting
amino acid interactions. Based on the experimental results and the model, we propose that a
salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce
electrostatic repulsion against
cation permeation caused by the positive residue(s). This
salt bridge may help stabilize the transporter configuration, and may also prevent the
conformational change that occurs in channels.
