The patch-damp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of Ki in asymmetric KCI solutions and also reversed near 0 mV at symmetric NaCl vs. KCI solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells.K1 killer toxin is a secreted heterodimeric protein produced by strains of Saccharomyces cerevisiae that contain an Ml virus with a double-stranded RNA genome (for a review, see ref. 1). The K1 killer toxin kills sensitive yeast cells lacking the Ml virus, whereas Ml virus-containing cells are immune to the toxin they produce. Toxin production and specific immunity to the toxin are encoded on the Ml double-stranded RNA component of the viral genome (2-5).The secreted toxin is processed from a larger precursor molecule that consists of an N-terminal leader, followed by the two toxin subunits, a and P which are separated by a central glycosylated y peptide (ref. 4 and for a review, see ref.6). Analysis of the predicted amino acid sequence of preprotoxin shows the a subunit possessing two highly hydrophobic regions (residues 72-91 and 112-127) separated by a short highly hydrophilic stretch of amino acids. It has been suggested that the hydrophobic a subunit is involved in ionchannel formation (4). The 13 subunit, however, lacks potential membrane-spanning regions. By analogy to the abrin and ricin class of toxins (7), this jS subunit has been proposed to function by binding to a cell wall receptor required for toxin action (4). Previous genetic and biochemical studies of Kikiller-toxin action on sensitive yeast cells indicate a set of specific cell surface interactions. These include binding to a (1 --6)-p-D-glucan-containing cell wall receptor (8, 9). After wall binding, energy-dependent processes result in lethal physiological changes. Ion leakage at the plasma membrane appears to be a primary cause for the lethality. In metabolically active cells, a rapid inhibition of net proton pumping from the cells, along with an inhibition of K+ and amino acid uptake, upon exposure to the killer toxin has been reported (10). F...

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 Ni 2؉ to the YggB-His 6 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-His 6 oligomers are more stable in the presence of Ni 2؉ , providing evidence that Ni 2؉ is coordinated between C termini from different subunits of the channel. Intersubunit coordination of Ni 2؉ 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. Mechanosensitive (MS)1 ion channels open upon membrane tension, and therefore they represent the simplest mechanosensors. MS channels have been implicated in many physiological processes from growth and cell volume regulation to hearing, blood pressure regulation, and pain sensation (reviewed in Ref. 1). Bacterial MS channels protect these cells against hypoosmotic shock. Two types of MS channels from the cytoplasmic membrane of Escherichia coli, MscL and MscS, play an essential role in the physiology of this bacterium, allowing the efflux of solutes from the cytoplasm when osmolarity of the external medium decreases (2-4). MscL, the large conductance MS channel, has been cloned (5), and a quaternary structure of its closed conformation has been determined (6). Based on this structure and the analysis of the channel gating, the open conformation has been predicted (7, 8) and experimentally confirmed (9, 10). Functional homologues of this channel have been found in other bacteria (11) and Archea (12), and structurally related protein from Neurospora has been also reported (13). The functional channel is a pentamer, and each subunit consists of two ␣-helical membrane-spanning domains TM1 and TM2 with both the C and N termini located in the cytoplasm (6). TM1s line the pore, and their hydrophobic residues form the primary, transmembrane gate (6, 14). It is postulated that there are two gates involved in the opening of the channel: the transmembrane and the cytoplasmic gates (7, 8) acting in accordance (15). The transmembrane gate is proposed to act as a pressure sensor, and upon application of pressure, this gate permits initial expansion of the channel without its full opening (7,8,10). It is proposed that the other, cytoplasmic gate, which allows full activation of the channel, is composed of five ␣-helical S1 segments of the cytoplasmic N termini being connected with TM1s via flexible linkers. According to the model, the applied pressure is transmitted to...

Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses, which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude, providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation. Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated protein.

MscS is a bacterial mechanosensitive channel that shows voltage dependence. The crystal structure of MscS revealed that the channel is a homoheptamer with a large chamber on the intracellular site. Our previous experiments indicated that the cytoplasmic chamber of the channel is not a rigid structure and changes its conformation upon the channel activation. In this study, we have applied various sized cosolvents that are excluded from protein surfaces. It is well known that such cosolvents induce compaction of proteins and prevent thermal fluctuations. It is also known that they shift channel equilibrium to the state of lower volume. We have found that large cosolvents that cannot enter the channel interior accelerate channel inactivation when applied from the cytoplasmic side, but they slow down inactivation when applied from the extracellular side. We have also found that small cosolvents that can enter the channel cytoplasmic chamber prevent the channel from opening, unlike the large ones. These data support our idea that the channel cytoplasmic chamber shrinks upon inactivation but also give new clues about conformational changes of the channel upon transitions between its functional states.