Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.

Structural studies have demonstrated that the extracellular L3 loop of porin constricts the channel and suggest that this loop might be involved in channel selectivity and gating. We previously showed that positively charged polyamines can induce changes in porin gating kinetics by stabilization of closed states. Here we report the effects of the mutation of two different aspartate residues of Escherichia coli OmpC porin on the polyamine sensitivity of the channel. Aspartate 105 or aspartate 118 on the L3 loop was replaced by glutamine by site-directed mutagenesis. The gating activity of the wild-type and mutant channels were studied by patch-clamp of liposomes containing reconstituted outer membrane fractions, in the absence or the presence of either polyamine spermine or cadaverine. Porin channels with a D118Q mutation, at the root of L3, still showed some, albeit milder, sensitivity to polyamine modulation. On the other hand, the D105Q mutation, at the tip of L3, abolished the increase in closing frequency which is typically observed in the presence of polyamines. We conclude that aspartate 105 primarily, but not aspartate 118, plays an important role in mediating the polyamine-induced changes in gating kinetics that result in the inhibition of the OmpC channel.

Porins are trimers of beta-barrels that form channels for ions and other hydrophilic solutes in the outer membrane of Gram-negative bacteria. The X-ray structures of OmpF and PhoE show that each monomeric pore is constricted by an extracellular loop that folds into the channel vestibule, a motif that is highly conserved among bacterial porins. Electrostatic calculations have suggested that the distribution of ionizable groups at the constriction zone (or eyelet) may establish an intrinsic transverse electrostatic field across the pore, that is perpendicular to the pore axis. In order to study the role that electrostatic interactions between pore residues may have in porin function, we used spontaneous mutants and engineered site-directed mutants that have an altered charge distribution at the eyelet and compared their electrophysiological behavior with that of wild-type OmpC. We found that some mutations lead to changes in the spontaneous gating activity of OmpC porin channels. Changes in the concentration of permeant ions also altered this activity. These results suggest that the ionic interactions that exist between charged residues at the constriction zone of porin may play a role in the transitions between the channel's closed and open states.

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By use of the patch clamp technique, we have compared the electrophysiological signature of OmpC porin channels at neutral and acidic pH.

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