DISRUPTION OF POLYAMINE MODULATION BY A SINGLE AMINO-ACID SUBSTITUTION ON THE L3 LOOP OF THE OMPC PORIN CHANNEL

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