Functional Characterization of the KtrAB Potassium Transport System

Abstract

The Ktr transport system belongs to the large superfamily of Trk/Ktr/HKT ion transporters that share structural similarities with potassium and sodium channels. The KtrAB complex is composed by a dimer of the membrane-integrant KtrB and an octameric ring of the cytosolic KtrA protein, which is an RCK-domain. Each KtrB subunit has a K+ selective pore. We tested several nucleotides for eluting KtrA from a high-affinity resin and found that ADP and ATP are the most potent suggesting higher affinities towards the protein. The crystal structure of the complex, recently solved by our group, revealed that the ring conformation of KtrA changes upon nucleotide binding. To determine the functional effects of these nucleotides we reconstituted the KtrAB complex from B. subtilis into liposomes and measured the transport activity reflected by uptake of 86Rb+. Our results show that ATP activates the transporter, while the flux mediated by KtrAB-ADP is similar to the flux of KtrB reconstituted without KtrA. Increasing amounts of extraliposomal K+ or Na+ showed that Na+ permeates through the transporter, yet KtrAB favors K+ over Na+. In addition, the cytosolic ring regulates the activity of the transmembrane pore, however, it does not render selectivity towards ions as there are no selectivity differences between the KtrAB complex, either with ADP or ATP, and KtrB alone. In conclusion, our data shows that KtrA regulates the activity of KtrB and raises new ideas about the mechanism of activation by RCK domains in the superfamily. Current investigation attempts to characterize the molecular basis of activation.