Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli.

Abstract

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions ofa lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation

“…4). Two regions of localized conservation were noted, shown previously to encompass transmembrane elements (Blount et al, 1996b). The cytoplasmic ends of these segments, corresponding to the E. coli regions G14-F29 and V72-F85, bear the highest degree of conservation.…”

“…In the direction of translation, the MCS contains one consensus each for XhoI, Not I, Bgl II, Sal I and XbaI. E. coli strain PB104 (Blount et al, 1996b) was used to host the intermediate cloning steps and the expression constructs. Cultures were grown at 37ЊC in Luria-Bertani (LB) medium plus ampicillin (100 g ml ¹1 ).…”

“…Thus, MscL, as the first key to the mystery of mechanosensory transduction, presents a simple, accessible system for assessing how tension in a lipid membrane can affect the gating of an MS channel (Blount et al, 1996b;Hä se et al, 1997a,b;Sukharev et al, 1997).…”

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

“…12 Many studies have been performed using site-directed mutagenesis in order to better understand the structure-function of MscL. [13][14][15][16][17][18][19] One of these studies produced an estimation of the tension-sensing site in MscL using random scanning mutagenesis, where individual hydrophobic AAs facing the lipids were replaced with the hydrophilic AA asparagine to identify any "loss-of-function" mutants lacking mechanosensitivity. 15 As the result, it was found that replacement of one of seven amino residues located at the periplasmic end of the transmembrane helices caused the loss of MscL mechanosensitivity, suggesting that one or some of them may act as a tension sensor in MscL.…”

Conformational pathways in the gating of Escherichia coli mechanosensitive channel

“…MS channels are gated channels that stay in the closed state and open up when there is a tension in the cell membrane (6)(7)(8)(9)(10)(11)(12)(13)(14). The only available crystal structure is that of TbMscL (15), which is a homopentamer, with each monomer composed of a transmembrane domain and a cytoplasmic domain.…”