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Biophys J, August 2000, p. 802-813, Vol. 79, No. 2

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

Michael Schrader,* Klaus Fendler,* Ernst Bamberg,* Michael Gassel,dagger Wolfgang Epstein,Dagger Karlheinz Altendorf,dagger and Stefan Drösedagger

 *Max-Planck-Institut für Biophysik, D-60596 Frankfurt am Main, Germany;  dagger Universität Osnabrück, Fachbereich Biologie und Chemie, D-49069 Osnabrück, Germany; and  Dagger Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637 USA

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 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.

Biophys J, August 2000, p. 802-813, Vol. 79, No. 2
© 2000 by the Biophysical Society   0006-3495/00/08/802/12  $2.00


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