The trimeric protein known as porin, found in the outer membrane of Escherichia coli, forms ion channels which have been reported to be voltage-gated. The aim of this investigation is to increase the understanding of the function of such channels, particularly with respect to voltage-gating. To achieve this, porin was first extracted from whole cells of E. coli and then it was incorporated into lipid bilayers, made using the Montal-Mueller method, for subsequent investigation.
The extraction of porin from E. coli exploited both its tight association with
peptidoglycan and its high level of detergent resistance. Porin was extracted from two strains of E. coli, 0111 :B4 and K12 (a rough mutant), with the resulting samples being rich in OmpF and Ompe porins respectively.
Measurement of ion flow through the porin channels was made with different
transmembrane voltages and with a variety of salts and salt concentrations. Such
measurements enabled the selectivity of the channels to specific ions to be determined and estimates of pore diameter to be made. Instantaneous IN characteristics were asymmetrical and non-linear for both porins and were influenced by the salt concentration, pH of the electrolyte and the number of channels in the bilayer. Based on electrostatic models of gramicidin-like channels, coupled with the available structural information on porin, it was concluded that the porinlLPS complex inserts unidirectionally into the bilayer and has a net negative charge at the LPS end. The influence of the number of channels in the bilayer on the IN characteristic, most marked for the 0111 :B4 porin, was interpreted as being due to 'clusters' of channels in close association an idea supported by the results of voltage-gating experiments.
Voltage-gating in single channels was shown to occur in three well defined
steps, consistent with the known trimeric structure of porin. Multi-channel membranes showed several different types of current relaxation in response to a sustained voltage application. The most common type was composed of two exponential decays, suggesting the presence of two populations of closing channels. The results from voltage-gating experiments were compared with the theoretical behaviour of a simple two-state channel. This model was also extended to include entropy changes and it was concluded that porin followed the general behaviour predicted by a simple twostate model.