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 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.
Liu
N
N
Department of Biology, University of Houston, TX 77204-5513, USA.
Benedik
M J
MJ
Delcour
A H
AH
D001224:Aspartic Acid / Q000235:genetics; D002103:Cadaverine / Q000494:pharmacology; D004594:Electrophysiology; D004926:Escherichia coli; D005973:Glutamine / Q000235:genetics; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011073:Polyamines / Q000378:metabolism / Q000494:pharmacology*; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013096:Spermine / Q000494:pharmacology
D008081:Liposomes; D011073:Polyamines; D018272:Porins; D005973:Glutamine; D013096:Spermine; D001224:Aspartic Acid; D002103:Cadaverine