Andrzej Kubalski

C Termini of the Escherichia coli Mechanosensitive Ion Channel (MscS) Move Apart upon the Channel Opening

Heptameric YggB is a mechanosensitive ion channel (MscS) from the inner membrane of Escherichia coli. We demonstrate, using the patch clamp technique, that cross-linking of the YggB C termini led to irreversible inhibition of the channel activities. Application of Ni2+ to the YggB-His6 channels with the hexahistidine tags added to the ends of their C termini also resulted in a marked but reversible decrease of activities. Western blot revealed that YggB-His6 oligomers are more stable in the presence of Ni2+, providing evidence that Ni2+ is coordinated between C termini from different subunits of the channel. Intersubunit coordination of Ni2+ affecting channel activities occurred in the channel closed conformation and not in the open state. This may suggest that the C termini move apart upon channel opening and are involved in the channel activation. We propose that the as yet undefined C-terminal region may form a cytoplasmic gate of the channel. The results are discussed and interpreted based on the recently released quaternary structure of the channel.

Genetic Screen for Potassium Leaky Small Mechanosensitive Channels (MscS) in Escherichia coli RECOGNITION OF CYTOPLASMIC β DOMAIN AS A NEW GATING ELEMENT

Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore.

GLUTATHIONE (GSH) REDUCES THE OPEN PROBABILITY OF MECHANOSENSITIVE CHANNELS IN ESCHERICHIA COLI PROTOPLASTS

Abstract The effects of glutathione (reduced GSH, and oxidized GSSG) on mechanosensitive (MS) ion channels from Escherichia coli protoplasts were investigated using excised, inside-out membrane patches. Our studies demonstrate here that 5 mM GSH irreversibly reduces the activities of the 560-pS MS channel by approximately 70–75% whereas 5 mM GSSG did not alter the MS channel currents. In addition, millimolar concentrations of dithiothreitol had similar although weaker effects to GSH. The physiological concentration of GSH in E. coli cytoplasm ranges from 3.5 mM to 6.6 mM, which may indicate that under normal conditions these MS channels open less due to membrane stretch.

Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

The MscS Cytoplasmic Domain and Its Conformational Changes on the Channel Gating

Publisher Summary The cytoplasmic domain of the bacterial mechanosensitive (MS) channel of small conductance (MscS) is shaped by its C‐termini forming a large chamber filled with water. Studies indicate that the chamber is a dynamic structure that undergoes severe conformational changes on the channel gating. Various electrophysiological and biochemical methods combined with molecular biology have been used to investigate this phenomenon and the results are presented in this chapter. The size of the chamber and its shape resemble cytoplasmic domains from eukaryotic non‐MS channels whose function in stabilization of the channel closed state is established. Analogous role of the MscS cytoplasmic chamber is discussed. Bacterial MS channels protect these cells against hypoosmotic shock. Two types of MS channels from the cytoplasmic membrane of Escherichia coli , MscL and MscS (the large and small conductance MS channel, respectively), play an essential role in the physiology of this bacterium, allowing efflux of solutes from the cytoplasm when osmolarity of the external medium decreases.

Negative and positive temperature dependence of potassium leak in MscS mutants: Implications for understanding thermosensitive channels.

Bacterial mechanosensitive channel of small conductance (MscS) is a protein, whose activity is modulated by membrane tension, voltage and cytoplasmic crowding. MscS is a homoheptamer and each monomer consists of three transmembrane helices (TM1-3). Hydrophobic pore of the channel is made of TM3s surrounded by peripheral TM1/2s. MscS gating is a complex process, which involves opening and inactivation in response to the increase of membrane tension. A number of MscS mutants were isolated. Among them mutants affecting gating have been found including gain-of-function (GOF) and loss-of-function (LOF) that open at lower or at higher thresholds, respectively. Previously, using an in vivo screen we isolated multiple MscS mutants that leak potassium and some of them were GOF or LOF. Here we show that for a subset of these mutants K+ leak is negatively (NTD) or positively (PTD) temperature dependent. We show that temperature reliance of these mutants does not depend on how MS gating is affected by a particular mutation. Instead, we argue that NTD or PTD leak is due to the opposite allosteric coupling of the structures that determine the temperature dependence to the channel gate. In PTD mutants an increased hydration of the pore vestibule is directly coupled to the increase in the channel conductance. In NTD mutants, at higher temperatures an increased hydration of peripheral structures leads to complete separation of TM3 and a pore collapse.