Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis

Rasmussen, Tim; Rasmussen, Akiko; Singh, Shivani; Galbiati, Heloisa; Edwards, Michelle D.; Miller, Samantha; Booth, Ian R.

doi:10.1021/acs.biochem.5b00294

Cited by 20 publications

(16 citation statements)

References 68 publications

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“…3 C). More interestingly, mutation of the key residues that gate the non-selective Ec MscS is consistent with our findings that the Ec MscS L105M ( Ec MscS L105 corresponds to the YnaI M158 ) obtains cation selectivity compared with the wild type (Bass et al, 2002 ; Rasmussen et al, 2015 ), while the Ec MscS L109M ( Ec MscS L109 corresponds to the YnaI K161 ) mutation has no effect on the ion selectivity (Figs. 3 D and S5).…”

Section: Resultssupporting

confidence: 91%

A binding-block ion selective mechanism revealed by a Na/K selective channel

Zhang

et al. 2017

Protein &Amp; Cell

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Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na+/K+ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaIMet158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaIMet158 facilitate Na+/K+ pass through, which was defined as binding-block mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0465-8) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 91%

A binding-block ion selective mechanism revealed by a Na/K selective channel

Zhang

et al. 2017

Protein &Amp; Cell

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“…While these proteins are diverse in structure and function, they may not fully reflect the mutational propensities of other proteins. For example, tryptophan scanning mutagenesis is often applied to transmembrane domains ( Sharp et al 1995 ; Depriest et al 2011 ; Rasmussen et al 2015 ), which were absent from the proteins we analyzed. Thus, our conclusions are most applicable to soluble proteins.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Large-Scale Mutagenesis Data To Assess the Impact of Single Amino Acid Substitutions

2017

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Mutagenesis is a widely used method for identifying protein positions that are important for function or ligand binding. Advances in high-throughput DNA sequencing and mutagenesis techniques have enabled measurement of the effects of nearly all possible amino acid substitutions in many proteins. The resulting large-scale mutagenesis data sets offer a unique opportunity to draw general conclusions about the effects of different amino acid substitutions. Thus, we analyzed 34,373 mutations in 14 proteins whose effects were measured using large-scale mutagenesis approaches. Methionine was the most tolerated substitution, while proline was the least tolerated. We found that several substitutions, including histidine and asparagine, best recapitulated the effects of other substitutions, even when the identity of the wild-type amino acid was considered. The effects of histidine and asparagine substitutions also correlated best with the effects of other substitutions in different structural contexts. Furthermore, highly disruptive substitutions like aspartic and glutamic acid had the most discriminatory power for detecting ligand interface positions. Our work highlights the utility of large-scale mutagenesis data, and our conclusions can help guide future single substitution mutational scans.

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“…The closed form of Ec MscS is proposed to involve close packing of small residues from adjoining pore-lining domains (Edwards et al, 2005). The substitution of large hydrophobic residues for Gly or Ala at these positions results in less stable open state configurations with higher tension thresholds, that at least superficially resemble MSL8 F720L (Edwards et al, 2005; Rasmussen et al, 2015; Wu et al, 2011). Alternating chains of small hydrophobic amino acids are not observed in the predicted pore-lining domain of MSL8 (Fig.…”

Section: Discussionmentioning

confidence: 99%

The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination

Hamilton¹,

Haswell

2016

Preprint

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All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in a msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild type Ler background, MSL8I711S suppressed pollen germination, similar to wild type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required to for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer.

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Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis

Cited by 20 publications

References 68 publications

A binding-block ion selective mechanism revealed by a Na/K selective channel

A binding-block ion selective mechanism revealed by a Na/K selective channel

Analysis of Large-Scale Mutagenesis Data To Assess the Impact of Single Amino Acid Substitutions

The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination

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