PMID:10920013
| Citation |
Schrader, M, Fendler, K, Bamberg, E, Gassel, M, Epstein, W, Altendorf, K and Dröse, S (2000) Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex. Biophys. J. 79:802-13 |
|---|---|
| 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 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. |
| Links |
PubMed PMC1300979 Online version:10.1016/S0006-3495(00)76337-5 |
| Keywords |
Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/metabolism; Amino Acid Sequence; Amino Acid Substitution; Aspartic Acid; Carrier Proteins/chemistry; Carrier Proteins/metabolism; Cation Transport Proteins; Cell Membrane/physiology; Cell Membrane/ultrastructure; Escherichia coli/physiology; Escherichia coli Proteins; Glycine; Kinetics; Lipid Bilayers; Liposomes; Macromolecular Substances; Membrane Potentials; Models, Molecular; Molecular Sequence Data; Potassium/metabolism; Protein Conformation; Proteolipids/chemistry; Proteolipids/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism |
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