Replacement of Glycine 232 by Aspartic Acid in the KdpA Subunit Broadens the Ion Specificity of the K +-Translocating KdpFABC Complex

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 (Buurman et al., 1995, 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 (Jan and Jan, 1994, Nature. 371:119–122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC 3(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, DiSC 3(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.