Mutagenesis identifies amino acid residues in extracellular loops and within the barrel lumen that determine voltage gating of porin from Haemophilus influenzae type b.
Porin (341 amino acids; M(r) 37 782) of Haemophilus influenzae type b mediates exchange of solutes between the external environment and the periplasm of this Gram-negative bacterium. Positively charged residues in the extracellular loops have been shown to be involved in the voltage gating of this protein. To further elucidate our observations on the functional properties of this channel, we mutated seven lysines (Lys(48), Lys(161), Lys(165), Lys(170), Lys(248), Lys(250), and Lys(253)) to glutamic acid. The selected residues were previously shown to be accessible to chemical modification, and they map to three locations: loop 4 and loop 6, and within the barrel lumen. The seven mutant proteins were purified, and each was reconstituted into planar lipid bilayers to characterize its channel forming properties. The single substitution mutant porins displayed increased single channel conductances in 1 M KCl ranging between 134 and 178% of the single channel conductance for wild-type Hib porin. Six of the seven mutant porins also displayed altered current-voltage relationships when compared to wild-type Hib porin. Whereas Lys(170)Glu had activity similar to wild-type Hib porin, Lys(48)Glu, Lys(248)Glu, and Lys(253)Glu showed substantial voltage gating at both positive and negative polarities. Lys(161)Glu and Lys(250)Glu gated only at negative potentials, and Lys(165)Glu gated only at positive potentials. Rather than ascribing one specific loop in gating, our analyses of these mutant Hib porins suggest that voltage gating can be attributed to contributions from loops 4 and 6 and a residue within the barrel lumen.
Arbing
M A
MA
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada.
Hanrahan
J W
JW
Coulton
J W
JW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002463:Cell Membrane Permeability / Q000502:physiology; D017931:DNA Primers / Q000737:chemistry; D004553:Electric Conductivity; D019741:Haemophilus influenzae type b / Q000737:chemistry / Q000235:genetics*; D015640:Ion Channel Gating / Q000502:physiology*; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary
D017931:DNA Primers; D008051:Lipid Bilayers; D018272:Porins