The zinc binding site of the Shaker channel KDC1 from Daucus carota.
KDC1 is a voltage-dependent Shaker-like potassium channel subunit cloned from Daucus carota which produces conductive channels in Xenopus oocytes only when coexpressed with other plant Shaker potassium subunits, such as KAT1 from Arabidopsis thaliana. External Zn(2+) determines a potentiation of the current mediated by the dimeric construct KDC1-KAT1, which has been ascribed to zinc binding at a site comprising three histidines located at the S3-S4 (H161, H162) and S5-S6 (H224) linkers of KDC1. Here we demonstrate that also glutamate 164, located in close proximity of the KDC1 S4 segment, is an essential component of the zinc-binding site. On the contrary, glutamate 159, located in symmetrical position with respect to E164 in the sequence E(159)XHHXE(164) but more distant from the voltage sensor, does not play any role in zinc binding. The effects of Zn(2+) can be expressed as a "shift" of the gating parameters along the voltage axis. Kinetic modeling shows that Zn(2+) slows the closing kinetics of KDC1-KAT1 without affecting the opening kinetics. Possibly, zinc affects the movement of the voltage sensor in and out of the membrane phase through electrostatic modification of a site close to the voltage sensor.
Picco
Cristiana
C
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genoa, Italy. picco@ge.ibf.cnr.it
Naso
Alessia
A
Soliani
Paolo
P
Gambale
Franco
F
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001665:Binding Sites; D018552:Daucus carota / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D014982:Xenopus laevis; D015032:Zinc / Q000378:metabolism*
C419388:KDC1 protein, Daucus carota; D050505:Mutant Proteins; D010940:Plant Proteins; D015221:Potassium Channels; D018698:Glutamic Acid; D007811:Lanthanum; D015032:Zinc