Voltage gating of Escherichia coli porin channels: role of the constriction loop.
In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the beta-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the high-resolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 A. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C/A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (Delta116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.
Phale
P S
PS
Department of Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Schirmer
T
T
Prilipov
A
A
Lou
K L
KL
Hardmeyer
A
A
Rosenbusch
J P
JP
D004594:Electrophysiology; D004926:Escherichia coli / Q000502:physiology* / Q000648:ultrastructure; D015640:Ion Channel Gating; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014961:X-Ray Diffraction
D018272:Porins