Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin

The porin from Paracoccus denitrificans, a slightly anion specific outer me mbrane pore protein, was expressed in Escherichia coli, isolated from inclu sion bodies, and refolded in the presence of urea and detergents. The purif ied recombinant protein was reconstituted into black lipid bilayer membrane s and showed no difference in its functional properties in comparison to th e native porin isolated from P. denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of sit e-directed mutants was constructed, comprising acidic residues located on t he third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements us ing zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-depende nt closure of channels, only mutations located on the L3 loop showed an eff ect, in contrast to the voltage-independent recombinant and native Paracocc us porin.