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

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Schrader,  Michael
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Fendler,  Klaus
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Bamberg,  Ernst
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Schrader, M., Fendler, K., Bamberg, E., Gassel, M., Epstein, W., Altendorf, K., et al. (2000). Replacement of Glycine 232 by Aspartic Acid in the KdpA Subunit Broadens the Ion Specificity of the K+-translocating KdpFABC Complex. Biophysical Journal, 79(2), 802-813. doi:10.1016/S0006-3495(00)76337-5.


Cite as: http://hdl.handle.net/21.11116/0000-0007-D2B5-F
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.