Molecular basis of plant-specific acid activation of K+ uptake channels.
During stomatal opening potassium uptake into guard cells and K+ channel activation is tightly coupled to proton extrusion. The pH sensor of the K+ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K+ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K+ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K+ uptake channels.
Hoth
S
S
Institut für Biophysik, Universität Hannover, Herrenhäuser Strasse 2, 30419 Hannover, Germany.
Dreyer
I
I
Dietrich
P
P
Becker
D
D
Müller-Röber
B
B
Hedrich
R
R
D001216:Asparagine / Q000502:physiology; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006639:Histidine / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D018515:Plant Leaves / Q000166:cytology / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D017494:Proton Pumps; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D011198:Solanum tuberosum; D013045:Species Specificity
D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D017494:Proton Pumps; D011994:Recombinant Proteins; D006639:Histidine; D001216:Asparagine