The pore of plant K(+) channels is involved in voltage and pH sensing: domain-swapping between different K(+) channel alpha-subunits.
Plant K(+) uptake channel types differ with respect to their voltage, Ca(2)+, and pH dependence. Here, we constructed recombinant chimeric channels between KST1, a member of the inward-rectifying, acid-activated KAT1 family, and AKT3, a member of the weakly voltage-dependent, proton-blocked AKT2/3 family. The homologous pore regions of AKT3 (amino acids 216 to 287) and KST1 (amino acids 217 to 289) have been exchanged to generate the two chimeric channels AKT3/(p)KST1 and KST1/(p)AKT3. In contrast to AKT3 wild-type channels, AKT3/(p)KST1 revealed a strong inward rectification reminiscent of that of KST1. Correspondingly, the substitution of the KST1 by the AKT3 pore led to less pronounced rectification properties of KST1/(p)AKT3 compared with wild-type KST1. Besides the voltage dependence, the interaction between the chimera and extracellular H(+) and Ca(2)+ resembled the properties of the inserted rather than the respective wild-type pore. Whereas AKT3/(p)KST1 was acid activated and Ca(2)+ insensitive, extracellular protons and Ca(2)+ inhibited KST1/(p)AKT3. The regulation of the chimeric channels by cytoplasmic protons followed the respective wild-type backbone of the chimeric channels, indicating that the intracellular pH sensor is located outside the P domain. We thus conclude that essential elements for external pH and Ca(2)+ regulation and for the rectification of voltage-dependent K(+) uptake channels are located within the channel pore.
Hoth
S
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Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
Geiger
D
D
Becker
D
D
Hedrich
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D014982:Xenopus laevis
D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D002118:Calcium