ABSTRACT
The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.
Subject(s)
Bacterial Proteins/chemistry , Corynebacterium glutamicum/chemistry , Escherichia coli/chemistry , Ion Channels/chemistry , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Corynebacterium glutamicum/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Ion Channel Gating , Ion Channels/genetics , Ion Channels/metabolism , Liposomes/chemistry , Liposomes/metabolism , Mechanotransduction, Cellular , Membrane Potentials/physiology , Molecular Sequence Data , Patch-Clamp Techniques , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spheroplasts/chemistry , Spheroplasts/genetics , Spheroplasts/metabolism , Structure-Activity RelationshipABSTRACT
Liposomal drug delivery systems (LDDSs) are promising tools used for the treatment of diseases where highly toxic pharmacological agents are administered. Currently, destabilising LDDSs by a specific stimulus at a target site remains a major challenge. The bacterial mechanosensitive channel of large conductance (MscL) presents an excellent candidate biomolecule that could be employed as a remotely controlled pore-forming nanovalve for triggered drug release from LDDSs. In this study, we developed superparamagnetic nanoparticles for activation of the MscL nanovalves by magnetic field. Synthesised CoFe2O4 nanoparticles with the radius less than 10 nm were labelled by SH groups for attachment to MscL. Activation of MscL by magnetic field with the nanoparticles attached was examined by the patch clamp technique showing that the number of activated channels under ramp pressure increased upon application of the magnetic field. In addition, we have not observed any cytotoxicity of the nanoparticles in human cultured cells. Our study suggests the possibility of using magnetic nanoparticles as a specific trigger for activation of MscL nanovalves for drug release in LDDSs.
