Disruption of polyamine modulation by a single amino acid substitution on the L3 loop of the OmpC porin channel

Abstract

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.

Complex Inhibition of OmpF and OmpC Bacterial Porins by Polyamines

“…2, B and D, shows that spermine promotes also a widening of the foot of the base line in the positive range because of an increase in the number of closing transitions involving one or two channels. When the number and duration of closures become large enough, distinct peaks of positive values can be seen on the amplitude histograms (19). These effects are observed also with spermidine and cadaverine (but not putrescine) and are better documented with the use of an analysis algorithm based on event detection, as described below.…”

“…These two types of activities most likely represent channels that occupy different stable conformational states that are interconvertible. We know that they originate from a single type of channel because both the closing and opening kinetics from the base-line level can be affected by single amino acid mutations of the OmpC protein (19,24). In addition, opening transitions are always more clearly observable at positive pipette potentials than negative ones and more so in OmpC than in OmpF.…”

“…The decreased occurrence of openings in the presence of polyamines is reflected in the amplitude histograms by the disappearance of the left shoulder and the concomitant narrowing of the base-line peak in the negative range. This effect can be measured quantitatively by comparing the abscissa of points that have the same ordinate value of 500 points/bin and are taken from the two curves (19). This parameter (I 500 ) will be larger in conditions where numerous unresolved openings were observed.…”

“…In this report, we have extended our investigations to the modulation of homotrimers of OmpF or OmpC by all four polyamines (spermine, spermidine, cadaverine, and putrescine) and showed that the effects are complex. In conjunction with our previous work on polyamine inhibition of site-directed OmpC mutants (19), the present study allows us to present a model for the possible molecular interactions between porins and polyamines.…”

“…Thus, kinetic analysis was done only for the closures with an algorithm that uses the halfamplitude criterion (19) to classify events lasting for more than 300 s as closures of 1, 2, . .…”