The antibiotic protein colicin E1 forms ion channels in planar lipid b
ilayers that are capable of conducting monovalent organic cations havi
ng mean diameters of at least 9 Angstrom. Polyvalent organic cations a
ppear to be completely impermeant, regardless of size. All permeant io
ns, whether large or small, positively or negatively charged, are cond
ucted by this channel at very slow rates. We have examined the permeab
ility of colicin E1 channels to anionic probes having a variety of siz
es, shapes, and charge distributions. In contrast to the behavior of c
ations, polyvalent as well as monovalent organic anions were found to
permeate the colicin E1 channel. Inorganic sulfate was able to permeat
e the channel only when the pH was 4 or less, conditions under which t
he colicin E1 protein is predominantly in an anion-preferring conforma
tional state. The less selective state(s) of the colicin E1 channel, o
bserved when the pH was 5 or greater, was not permeable to inorganic s
ulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N',N'-te
tramethyl-1,6-hexanediamine) had no effect on the single channel condu
ctance of colicin E1 channels exposed to solutions containing 1 M NaCl
at pH 5. The complete lack of blocking activity by either of these tw
o impermeant ions indicates that both are excluded from the channel lu
men. These results are consistent with our hypothesis that there is bu
t a single location in the lumen of the colicin E1 channel where posit
ively charged groups can be effectively hydrated. This site may coinci
de with the location of the energetic barrier which impedes the moveme
nt of anions.