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Abstract

Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K⁺-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K⁺ and has lost the ability to discriminate Rb⁺ ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K⁺ channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC₃(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K⁺ or Rb⁺, a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC₃(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na⁺, Li⁺, and H⁺ with a reduced rate. Similarities to mutations in the GYG motif of K⁺ channels are discussed.