Effects of GsMTx4 on bacterial mechanosensitive channels in inside-out patches from giant spheroplasts.
GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata folded into an inhibitory cysteine knot and it selectively affects gating of some mechanosensitive channels. Here we report the effects of cytoplasmic GsMTx4 on the two bacterial channels, MscS and MscL, in giant Escherichia coli spheroplasts. In excised inside-out patches, GsMTx4 sensitized both channels to tension by increasing the opening rate and decreasing the closing rate. With ascending and descending pressure ramps, GsMTx4 increased the gating hysteresis for MscS, a consequence of slower gating kinetics. Quantitative kinetic analysis of the primary C↔O transition showed that the hysteresis is a result of the decreased closing rate. The gating barrier location relative to the open state energy well was unaffected by GsMTx4. A reconstructed energy profile suggests that the peptide prestresses the resting state of MscS, lowering the net barrier to opening and stabilizes the open conformation by ∼8 kT. In excised patches, both MscL and MscS exhibit reversible adaptation, a process separable from inactivation for MscS. GsMTx4 decreased the rate of reversible adaptation for both channels and the MscS recovery rate from the inactivation. These measurements support a mechanism where GsMTx4 binds to the lipid interface of the channel, increasing the local stress that is sensed by the channels and stabilizing the expanded conformations.
Department of Biology, University of Maryland, College Park, Maryland, USA.
Kishore
K
Kamaraju
Philip A
PA
Gottlieb
Frederick
F
Sachs
Sergei
S
Sukharev
D000818:Animals; D055592:Biophysical Phenomena; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D036341:Intercellular Signaling Peptides and Proteins; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D013104:Spheroplasts / Q000187:drug effects / Q000378:metabolism; D013111:Spider Venoms / Q000737:chemistry / Q000494:pharmacology*
D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; C411249:MTx4 protein, Grammostola spatulata; C086596:MscL protein, E coli; C469542:MscS protein, E coli; D010455:Peptides; D013111:Spider Venoms