Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.
The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.
Abrams
C K
CK
Department of Physiology & Biophysics, Albert Einstien College of Medicine, Bronx, New York 10461.
Jakes
K S
KS
Finkelstein
A
A
Slatin
S L
SL
D001483:Base Sequence; D003087:Colicins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry*; D011189:Potassium Chloride / Q000494:pharmacology
D003087:Colicins; D007473:Ion Channels; D008567:Membranes, Artificial; D010743:Phospholipids; D011189:Potassium Chloride