VOLTAGE GATING OF PORINS FROM HAEMOPHILUS-INFLUENZAE TYPE-B

The major outer membrane protein of Haemophilus influenzae type b (Hib ) is porin (M(r) 37,782; 341 amino acids). Porins were purified from H ib strains representative of the three outer membrane protein subtypes 1H, 2L and 6U, reconstituted into artificial planar bilayers, and tes ted for their voltage dependency. At membrane potentials of 50-80 mV, individual Hib 2L and 6U porin channels showed a high probability of u ndergoing a reversible change to one of several lower conducting subst ates. Such behaviour was not observed for Hib 1H porin with transmembr ane potentials up to 80 mV. The voltage dependence of Hib 2L and 6U po rins was asymmetric: it occurred at only one polarity. The asymmetry w as also observed for membranes with numerous porins incorporated, sugg esting that Hib porin inserted asymmetrically into the bilayer. At mac roscopic levels the voltage gating reduced the conductance by 25-50%, implying that the channels closed only partially. Hib 2L porin differs from Hib 1H porin by the substitution Arg(166)Gln and Hib 6U porin di ffers from Hib 1H porin by substitutions at ten amino acids including the change Arg(166)Leu. We conclude that substitutions at Arg(166) res idue, which is localized to surface-exposed loop number four, are asso ciated with a lowered threshold potential for the voltage gating of Hi b porin. This surface-exposed loop may play some role in the conformat ional changes that occur during voltage gating.