The voltage‐dependent activity of Escherichia coli porins in different planar bilayer reconstitutions

Abstract

Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichiu coli porins in planar bilayers is still a matter of debate. In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outcr membrane vesicles, secondly as salt-extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl-polyoxyethylene (Octyl-POE). These methods represent the major approaches to porin isolation and purification. All three were reconstituted into Schindler-type bilayers. Detergent-solubilised OmpF was also reconstituted into Montal-Mueller-and Mueller-Rudin-type bilayers. In all cases voltage-dependent closing of OmpF was observed. Octyl-POE-extracted PhoE porin was similarly investigated in all three types of planar bilayer. Two membrane-formation techniques appeared genuinely to alter the voltage sensitivity of the porins they contained. Firstly, porins in membranes formed by the Montal-Mueller technique sometimes showed an increase in voltage sensitivity during the first 30 min after bilayer formation. Secondly, membranes formed by the Mueller-Rudin technique on thick polyethylene septa showed both poor solvent drainage and a significantly reduced porin voltage sensitivity. [4]. They showed that OmpF produced water-filled channels with limited selectivity between different ions and that the trimer conductance was around 2.0 nS in 1 M alkali metal chlorides. Bcnz and co-workers used the MuellerRudin ('black film' bilayer formation technique and showed that OmpF had an ohmic current/voltage relationship, indicating that the open state of these channels was not voltagesensitive. On the other hand, Schiiidler and Rosenbusch clearly demonstrated that the channels in their experiments could be closed by incrcasing the applied membranc potential difference. Since thcn published results on many porins have divided into two groups. One group, iiiostly using the socalled Schindler technique of bilaycr formation, show OmpF [5, 61, PhoE [7] and OmpC (our unpublished results) to be voltage-sensitive channels that close at potential differences above 100 mV. The other, much larger, group of publications shows porins in Mucller-Rudin bilayers to close only rarely and even then not because of an applied potential [l, 8 ~ 101 (for a review see [Ill).

Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch‐clamp

“…The voltage dependence of porins is still debated. One group of laboratories found that porins could be closed by increasing the membrane potential [5][6][7][8][9][10]. The kinetics of closure was slow, with events in the range of seconds.…”

Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting

“…By measuring the ion conductance in vitro, porin trimers were found to be able to exhibit at least two functional states, an open and a closed state, in response to changes in the transmembrane potential difference, known as "voltage gating", and the voltage above which the porin channel will be closed is called the critical voltage, Vc. Although existing data generally support voltage gating in vitro, there are no data to support voltage gating in vivo (25,27).…”

“…Currently known porins fall into two distinct groups: 16-stranded general diffusion porins such as the matrix porin OmpF that transports ions and small molecules (Ͻ600 Da) without much selectivity and 18-stranded specific porins such as maltoporin that have a precise selectivity for a defined substrate (15, 17, 19 -22). General porin trimers exhibit symmetrical or asymmetrical voltage gating when reconstituted into planar lipid bilayers in electrophysiological studies (23)(24)(25)(26)(27). By measuring the ion conductance in vitro, porin trimers were found to be able to exhibit at least two functional states, an open and a closed state, in response to changes in the transmembrane potential difference, known as "voltage gating", and the voltage above which the porin channel will be closed is called the critical voltage, Vc.…”

Altered voltage sensitivity of mutant OmpC porin channels

“…Formation of planar bilayers was as described previously [1,6] with the following exceptions. The solution used to coat the hole in the teflon septum across which the bilayer was formed, was 1% hexadecane in n-pentane and bilayers were formed using Schindler's technique [21].…”

“…These porins have been studied in detail in planar bilayers [1][2][3][4][5][6], showing each has a characteristic channel conductance and voltage-gating behaviour. Most porins have been observed to open and close in a controlled manner as a function of potential difference (pd).…”