Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure |
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Authors: | Trude M Haug Sverre L Sand Olav Sand Danh Phung Per E Granum Simon P Hardy |
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Institution: | (1) Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz, Austria;(2) School of Molecular and Systems Medicine, Alberta Diabetes Institute, University of Alberta, 6126 HRIF East, Edmonton, AB, T6G 2E1, Canada; |
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Abstract: | Mutation E71A in the bacterial K+-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide
backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A
by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either
in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular
partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular
TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and ~2.5-fold lower affinity
for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol
(PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel
conductance and increases the channel open time and open probability. Taken together, our data suggest that the “flipped”
conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding,
whereas higher PG content decreases channel conductivity and stabilizes the channel open “flipped” state via electrostatic
interaction in the proximity of the channel pore. |
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