KtrAB from Vibrio alginolyticus is a recently described new type of high affinity bacterial K+ uptake system. Its activity assayed in an Escherichia coli K+ uptake negative mutant depended on Na+ ions (Km of 40 microM). Subunit KtrB contains four putative P-loops. The selectivity filter from each P-loop contains a conserved glycine residue. Residue Gly-290 from the third P-loop selectivity filter in KtrB was exchanged for Ala, Ser or Asp. KtrB variants Ser-290 and Asp-290 were without activity. In contrast, KtrB variant Ala-290 was still active. This variant transported K+ with a two orders of magnitude decrease in apparent affinity for both K+ and Na+ with little effect on Vmax.