Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K+-translocating KdpFABC complex

Replacement of glycine residue 232 with aspartate in the KdpA subunit of th e K+-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K+ and has lost the ability to discr iminate Rbi ions (Buurman et at., 1995, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was alig ned with the GYG sequence of the selectivity filter (P- or H5-loop) of K+ c hannels (Jan and Jan, 1994, Nature. 371:119-122). investigations with the p urified and reconstituted KdpFABC complex using the potential sensitive flu orescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm t he 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 wi ld-type enzyme and with another kdpA mutant (A:Q116R) and most likely repre sents the movement of a protein-fixed charge during a conformational transi tion. After addition of K+ or Rb+, a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex, in add ition, DISC3(5) and planar bilayer measurements indicate that the A:G232D K dp-ATPase also transports Na+ Li+, and H+ with a reduced rate. Similarities to mutations in the GYG motif of K+ channels are discussed.