Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating.
Voltage-gated proton channels are strongly inhibited by Zn(2+), which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn(2+). We hypothesize that high-affinity Zn(2+) binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn(2+) effects were weaker in monomeric channels. Mutation of His(193) and His(140) in various combinations and in tandem dimers revealed that channel opening was slowed by Zn(2+) only when at least one His was present in each monomer, suggesting that in wild-type (WT) H(V)1, Zn(2+) binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn(2+) appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer-dimer interconversion occurs during activation of phagocytes appears to be ruled out.
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA.
Boris
B
Musset
Susan M E
SM
Smith
Sindhu
S
Rajan
Vladimir V
VV
Cherny
Sukrutha
S
Sujai
Deri
D
Morgan
Thomas E
TE
DeCoursey
D002460:Cell Line; D019281:Dimerization; D004594:Electrophysiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects* / Q000235:genetics; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D013696:Temperature; D014162:Transfection; D015032:Zinc / Q000494:pharmacology*
C510235:HVCN1 protein, human; D007473:Ion Channels; D049452:Green Fluorescent Proteins; D006639:Histidine; D015032:Zinc