Bacterial ion channels and their eukaryotic homologues.

Due to the relative ease of obtaining their crystal structures, bacterial ion channels provide a unique opportunity to analyse structure and function of their eukaryotic homologues. This review describes prokaryotic channels whose structures have been determined. These channels are KcsA, a bacterial homologue of eukaryotic potassium channels, MscL, a bacterial mechanosensitive ion channel and ClC0, a prokaryotic homologue of the eukaryotic ClC family of anion-selective channels. General features of their structure and function are described with a special emphasis on the advantages that these channels offer for understanding the properties of their eukaryotic homologues. We present amino-acid sequences of eukaryotic proteins related in their primary sequences to bacterial mechanosensitive channels. The usefulness of bacterial mechanosensitive channels for the studies on general principles of mechanosensation is discussed.

[Bacterial ion canals]. / Bakteryjne kanaly jonowe.

After an overview of potassium and chloride ion channels found in bacterial inner membrane this review focuses on mechanosensitive ion channels from the inner membrane of Escherichia coli. Mechanosensitive channels, MscL and MscS, have major roles in managing the transition from high to low environments. Biochemical and genetic studies of MscL, combined with structural information derived from X-ray crystalography, have brought the knowledge of how a mechanosensitive channel senses membrane tension. Physiological studies on MscL and on MscS have demonstrated how the mechanosensitive channels contribute to the bacterial membrane response upon hypo-osmotic stress.

Glutathione (GSH) reduces the open probability of mechanosensitive channels in Escherichia coli protoplasts.

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.

Voltage-independent adaptation of mechanosensitive channels in Escherichia coli protoplasts.

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.

Generation of giant protoplasts of Escherichia coli and an inner-membrane anion selective conductance.

Established methods were modified and combined to generate unilamellar giant protoplasts in order to study the electric events on the cytoplasmic membrane of Escherichia coli with patch-clamp technique. The activities of many types of conductances were encountered, one of them is characterized here. This channel conductance is 109 pS in 150 mM KCl; it prefers anions, it is highly voltage dependent and is blocked by micromolar concentrations of anthracene-9-carboxylic acid.

Activities of a mechanosensitive ion channel in an E. coli mutant lacking the major lipoprotein.

The activity of the mechanosensitive (MS) ion channels in membrane patches, excised from E. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wild-type parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the 1/Sp (the suction for an e-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities of lpp included a prominent substrate and showed a weaker mechanosensitivity with an 1/Sp of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in the lpp membranes. After lysolecithin addition, the lpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein services as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.

Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.

The patch-clamp 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 K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl 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.

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel in Paramecium.

The effects of proteolysis on a hyperpolarization- and Ca2+-dependent K channel from the surface membrane of Paramecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca2+-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca2+ concentrations between 10(-4) and 10(-8) M. Thermolysin additionally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca2+-dependent enzymes it is suggested that this Paramecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca2+ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca2+-dependent K channels (BK channels) and the hyperpolarization-, Ca2+-dependent K channels in Paramecium.