Probing sugar translocation through maltoporin at the single channel level

Abstract

Sugar permeation through maltoporin of Escherichia coli, a trimer protein that facilitates maltodextrin translocation across outer bacterial membranes, was investigated at the single channel level. For large sugars, such as maltohexaose, elementary events of individual sugar molecule penetration into the channel were readily observed. At small sugar concentrations an elementary event consists of maltoporin channel closure by one third of its initial conductance in sugar-free solution. Statistical analysis of such closures at higher sugar concentrations shows that all three pores of the maltoporin channel transport sugars independently. Interestingly, while channel conductance is only slightly asymmetric showing about 10% higher values at 3 3200 mV than at +200 mV (from the side of protein addition), asymmetry in dependence of the sugar binding constant on the voltage polarity is about 20 times higher. Combining our data with observations made with bacteriophage-V V we conclude that the sugar residence time is much more sensitive to (and is decreased by) voltages that are negative from the intra-cell side of the bacterial membrane. ß

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

“…4). Similar behavior has already been reported for the wild type maltoporin channels (17). The rate constants governing the open-close sequences experienced by a given channel can then be determined using Equation 2 by means of a linear plot of the reciprocal relaxation times versus maltohexaose concentrations.…”

“…The kinetics of maltohexaose binding through wild type maltoporin channels has been shown to be sensitive to the external voltage with higher influence on the apparent dissociation rate of bound substrates (17)(18)(19). This binding property has been also used to detect the directional insertion of wild type maltoporin.…”

“…Reconstitution experiments at the single-channel level allowed unambiguous exploration of the directional properties of maltoporin channels. These studies clearly showed that the channel is highly asymmetric with respect to the sugar entrance side and applied voltages and revealed that the maltoporin insertion in artificial membranes is always unidirectional (17)(18)(19). However, the real orientation of maltoporin remained an open question.…”

“…Recent conductance measurements performed on single maltoporin trimers show that the temporary binding of a maltodextrin molecule inside the channel can be observed as ion current fluctuation as penetrating sugar molecules constrain the passage of ions (17)(18)(19). Reconstitution experiments at the single-channel level allowed unambiguous exploration of the directional properties of maltoporin channels.…”

The Oligogalacturonate-specific Porin KdgM of Erwinia chrysanthemi Belongs to a New Porin Family

“…A well studied example is that of LamB (64,65), which facilitates the diffusion of maltose and maltodextrins. Although in this case, a well resolved block of the channel, at the single-channel level, can be observed (66), in the case of KdgM the short lived blocked states could not be resolved. At the very least, these data indicate that GA 3 is driven through the pore and that it interacts with the pore.…”