Histidines are responsible for zinc potentiation of the current in KDC1 carrot channels.
Unlike all plant inward-rectifying potassium channels, the carrot channel KDC1 has two histidine pairs (H161,H162) in the S3-S4 and (H224,H225) in the S5-S6 linkers. When coinjected with KAT1 in Xenopus oocytes, KDC1 participates in the formation of heteromultimeric KDC1:KAT1 channels and the ionic current is potentiated by extracellular Zn2+. To investigate the potential interactions between KDC1 and zinc, a KDC1-KAT1 dimer was constructed. The dimeric and heteromeric channels displayed similar characteristics and the same sensitivity to zinc and other metals; this result suggests that zinc binding is mediated by residues in a single channel subunit. The KDC1:KAT1 currents were also potentiated by external Pb2+ and Cd2+ and inhibited by Ni2+. To investigate further the role of KDC1-histidines, these amino acids were mutated into alanines. The single mutations H225A, H161A, and H162A did not affect the response of the heteromeric channels to zinc. Conversely, the single mutant H224A and the double mutants (H224A,H225A) and (H161A,H162A) abolished zinc potentiation, but not that induced by Pb2+ or Cd2+. These results suggest that Zn2+ potentiation cannot be ascribed to simple electrostatic interactions between zinc and channel residues and that histidine 224 is crucial for zinc but not for lead potentiation of the current.
Picco
Cristiana
C
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Sezione di Genova, Genova, Italy.
Bregante
Monica
M
Naso
Alessia
A
Gavazzo
Paola
P
Costa
Alex
A
Formentin
Elide
E
Downey
Patrick
P
Lo Schiavo
Fiorella
F
Gambale
Franco
F
D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D018552:Daucus carota / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008670:Metals / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010940:Plant Proteins / Q000819:agonists / Q000502:physiology*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D015221:Potassium Channels / Q000819:agonists / Q000502:physiology*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis / Q000235:genetics; D015032:Zinc / Q000494:pharmacology*
C419388:KDC1 protein, Daucus carota; D008670:Metals; D010940:Plant Proteins; D015221:Potassium Channels; D006639:Histidine; D015032:Zinc