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 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 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.
Schrader
M
M
Max-Planck-Institut für Biophysik, D-60596 Frankfurt am Main, Germany.
Fendler
K
K
Bamberg
E
E
Gassel
M
M
Epstein
W
W
Altendorf
K
K
Dröse
S
S
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D004926:Escherichia coli / Q000502:physiology; D029968:Escherichia coli Proteins; D005998:Glycine; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D001224:Aspartic Acid; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D011188:Potassium; D005998:Glycine