Chitoporin from the Marine Bacterium Vibrio harveyi

Background: VhChiP is a sugar uptake channel specific for chitohexaose. Results: Mutations of Trp136, located at the entrance of the transmembrane pore, affect ion and sugar transport through VhChiP. Conclusion: Trp136 regulates chitooligosaccharide uptake through VhChiP. Significance: Chitin uptake by the highly virulent bacterium V. harveyi through VhChiP is dependent on hydrophobic interactions between the sugar molecule and the channel surface. VhChiP is a sugar-specific porin present in the outer membrane of the marine bacterium Vibrio harveyi. VhChiP is responsible for the uptake of chitin oligosaccharides, with particular selectivity for chitohexaose. In this study, we employed electrophysiological and biochemical approaches to demonstrate that Trp136, located at the mouth of the VhChiP pore, plays an essential role in controlling the channel's ion conductivity, chitin affinity, and permeability. Kinetic analysis of sugar translocation obtained from single channel recordings indicated that the Trp136 mutations W136A, W136D, W136R, and W136F considerably reduce the binding affinity of the protein channel for its best substrate, chitohexaose. Liposome swelling assays confirmed that the Trp136 mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. Notably, all of the mutants show increases in the off-rate for chitohexaose of up to 20-fold compared with that of the native channel. Furthermore, the cation/anion permeability ratio Pc/Pa is decreased in the W136R mutant and increased in the W136D mutant. This demonstrates that the negatively charged surface at the interior of the protein lumen preferentially attracts cationic species, leading to the cation selectivity of this trimeric channel.

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