The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.
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.
Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia.
Yoshitaka
Y
Nakayama
,Institute of Biochemistry, University of Cologne, Zulpicher Str. 47, 50674 Koln, Germany.
Michael
M
Becker
Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia; Wollongong University, Sydney, NSW 2010, Australia.
Haleh
H
Ebrahimian
Department of Green and Sustainable Chemistry, Tokyo Denki University, 5 Asahi-cho, Senju, Adachi-ku, Tokyo 120-8551, Japan.
Tomoyuki
T
Konishi
Department of Green and Sustainable Chemistry, Tokyo Denki University, 5 Asahi-cho, Senju, Adachi-ku, Tokyo 120-8551, Japan.
Hisashi
H
Kawasaki
,Institute of Biochemistry, University of Cologne, Zulpicher Str. 47, 50674 Koln, Germany.
Reinhard
R
Krämer
Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia. Electronic address: b.martinac@victorchang.edu.au.
Boris
B
Martinac
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D048230:Corynebacterium glutamicum / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D040542:Mechanotransduction, Cellular; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013104:Spheroplasts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D007473:Ion Channels; D008081:Liposomes; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; C024990:asolectin
E. coli; Gating hysteresis; Glutamate efflux; Liposomes; Mechanosensitive channel; NCgl1221; Patch clamp; Spheroplasts