Microbial Channels

CHAPTER

29 Microbial Channels BORIS MARTINAC, XIN-LIANG ZHOU, ANDRZEJ KUBALSKI, SERGEI SUKHAREV, AND CHING KUNG

crobes. The challenge is in generating appropriate preparations so the microbial membranes can form gigaOhm seals with the patch-clamp pipettes. For example, Paramecium is completely covered with cilia; yeast membrane is covered by a cell wall, and different yeasts differ in their wall components; E. coli has two membranes, the outer and the inner membrane. No single method can be applied to all these situations. Figure 1 summarizes some of the methods used. Details of these methods can be found in Saimi et al. (1992) and in references cited for different organisms in the following discussion.

I. P a t c h - C l a m p Investigation of M i c r o b e s Microscopic creatures are vastly more diverse than plants and animals combined. These organisms represent different early evolutionary branches and consist of cells with very different body plans and metabolic styles. A formal classification of microbes is beyond the scope of this chapter. A reminder that the organisms we call microbes include prokaryotes and eukaryotes, among which are protists, algae, and fungi, should suffice. Primarily, this chapter addresses the activities of ion channels discovered in Paramecium, Dictyostelium, yeasts, and Escherichia coli. In addition to microbial biology and channel evolution, researchers have studied channel biology in molecular detail. Thus, most of the microbial species examined with patch-clamp techniques are those that are genetically tractable and have been used extensively in biochemical or molecular biological research. Prior to the advent of patch-clamp techniques, electrical measurements could only be made on giant cells in vivo or by incorporating microbial channels into planar lipid bilayers in vitro. The patch-clamp method not only allows examination of individual ion channels (Hamill et ai, 1982), but also allows the study of small cells. Little modification of the solutions, electrodes, and amplifiers is needed to record from miHANDBOOK

OF MEMBRANE

CHANNELS

II.

Paramecium

Although unicellular, Paramecium has many animal-like characteristics. This organism is a heterotroph and relies on dietary amino acids and vitamins. Paramecium swims about with 5000 incessantly beating cilia. Like those in higher animals, each cilium consists of a dynein-ATPase powered "9 + 2" assembly of sliding microtubules enclosed in a membrane. The Paramecium membrane is excitable, and ciliary motility is governed by membrane electrogenesis. The "avoidance reaction" (Jennings, 1906), for example, correlates with excitation. Depolarization can trigger a 2 + Ca -based action potential; the resultant intraciliary 447

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448

BORIS MARTINAC

STARTING MATERIAL

ET AL.

OBJECTS FOR PATCH CLAMP

PROCEDURES

ι

blister vesicles

I

^

^

Ο

detached cilia

spheroplasts

.© —

σ

2

©

mitochondria

Ο

vacuoles

= £ 3 » Q high osmo.

giant spheroplasts giant cells

Lpp'OmpA

giant cells

azolectin

azolectin

dere-hyd.

dere-hyd.

>

>

Mg

Mg

+

liposome blisters +

liposome blisters

FIGURE 1 Outline of procedures to generate microbial objects suitable for patch-clamp studies. The starting material (left)—Paramecium, budding yeast, Escherichia coli, membrane vesicles, and yeast killer toxin—have dimensions on the order of 100, 10, 1, 0.1, and 0.01 μιτι, respectively. The procedure (center), briefly stated here but detailed in Saimi et al. (1992) and references therein, converts them to objects used successfully in patch-clamp experiments. The objects (right) are all 5 - 1 5 μπ\ in diameter, except detached cilia from Paramecium ( ~ 2 μτη) and yeast mitochondria and vacuoles ( ~ 3 μτη). Paramecium blister vesicles + 2 + are induced in solutions of high [Na ] and low [ C a ] , and can be detached from the cell mechanically. These blisters have two membranes, the outer cytoplasmic membrane and the inner membrane, probably of alveolar origin. Detached cilia are sheared off paramecia forced through a narrow-bore pipet. Yeast spheroplasts are obtained after cell wall removal with zymolyase, an enzyme mixture from Athrobacter luteus. A hypo-osmotic treatment followed by osmotic re-adjustment can open the spheroplasts, each releasing a mitochondrion and a vacuole. These mitochondria and vacuoles are optically distinct and can be selected for patch-clamp studies. E. coli wild-type strain AW405 grows as filaments more than 100 μ,ιη long in the presence of cephalexin, a penicillin analog, or on exposure to ultraviolet light. These filaments round up into giant spheroplasts when treated with EDTA and lysozyme, which nicks the peptidylglycan cell wall. Giant cells are obtained by growing the wild-type or Lpp~Omp~ double-mutant cells, lacking 2+ the major outer-membrane anchors to the cell wall, in the presence of cephalexin and tens of m M [ M g ] . + 2+ About 10% of cells of the osmotic-sensitive mutant strain AW693 grown in high [K ] and millimolar [ M g ] are also giant cells. Vesicles of various microbial membrane fractions and yeast killer toxin are mixed with azolectin which, after a cycle of de- and rehydration, yields large multilamellar liposomes. Liposome blisters 2+ grow out of the liposomes after they have been collapsed by tens of m M [ M g ] . Objects are not drawn to scale. Reproduced with permission from Saimi et al. (1992).

29. Microbial Channels 2+

increase of C a causes the cilia to beat in a reversed direction to propel the cell backward (Naitoh and Eckert, 1969; Eckert et al, 1972). Hyperpolarization can trigger other ion currents and a more rapid ciliary beat in a near-normal direction, causing the cell to spurt forward (Naitoh and Eckert, 1969,1973). Under certain circumstances, hyperpolarization can become regenerative (Satow and Kung, 1977). Cyclic AMP is thought to play an important role in this spurting response (Hennessey et al., 1985; Bonini et al., 1986; Nakaoka and Machemer, 1990; Preston and Saimi, 1990). Schultz and co-workers (1992) suggested that, + in Paramecium, the K efflux underlying membrane hyperpolarization and cAMP increase might be affected by the same entity—the ciliary adenylate cyclase. The purified cyclase, on incorporation into planar lipid bilayers, was found to be associated with a 320-pS nonselective cation channel. Because the Paramecium cell is large, some 100 μιη in length, it has been investigated with the classical two-electrode voltage clamp as well as the patch clamp. Experiments with these two methods and with planar lipid bilayers revealed at least 13 types of ion C u r r e n t s : 7ca(d)/ Jca(h)/ A:a(mech)/ ^K(d)' ^K(h)/ ^K(Ca,d)' ^K(Ca,h)/ a n <

^ ^Μ + (ΑΤΡ)· ThlS large number of currents should not be surprising. First, this highly differentiated cell is anatomically complex. Although continuous, the plasma membrane and the ciliary membrane are distinct. Also, organelles are found in or attached to the plasma membrane, including an oral apparatus, a cytoproct, two contractile vacuoles, an alveolar-sac network, thousands of parasomal sacs, and trichocysts (Jurand and Selman, 1969). Second, as a free-living organism, Paramecium must move, respond, adapt, endocytose, excrete, secrete, divide, mate, and autogamize (Goertz, 1988). Some of these physiological functions are known or expected to have an ionic or electrical component. When Paramecium is examined bathed in various external solutions under a two-electrode voltage clamp, membrane depolarizations can trigger at least a 2+ five currents. (1) I Ca(d)/ depolarization-activated C a current, activates rapidly by voltage and inactivates 2+ in a Ca -dependent manner (Oertel et al., 1977; Brehm and Eckert, 1978). Deciliation abolishes this current, indicating that the channels are located in the ciliary membrane and not in the body membrane (Dunlap, 1977; Machemer and Ogura, 1979). "Pawn" mutations abolish or reduce this current (Kung and Eckert, 1972; Oertel et al, 1977; Satow and Kung, + 1980a). (2) 7 K ( d ), a rapid K current activated by depolarization, does not inactivate (Machemer and Ogura, + 1979; Satow and Kung, 1980a). (3) 7 N a ( C ,a )a N a cur2 + rent, is activated by internal C a , presumably from 2+ the already described C a current (Saimi and Kung, ^K(mech)/ ^Na(Ca)' ^Mg(Ca)/ ^Cl' ^M-M

449

1980; Saimi, 1986). A subclass of calmodulin mutants + called "fast-2" has little or none of this N a current (Kink et al, 1990; Preston et al, 1991; Ling et al, 1992; a + Kung et al, 1992). (4) 7K(Ca,d)> K current, is also 2+ activated by internal C a (Satow and Kung, 1980b; Preston et al, 1990b). A different subclass of calmodulin mutants called "pantophobiacs" shows large reductions in this current (Saimi et al, 1983; Kink et al, 1990; Preston et al, 1990b; Kung et al, 1992). This current is greatly enhanced in the tea A mutant (Hennessey and Kung, 1987; Preston et al, a 2+ 1990c). (5) / M (gC a ) ' Mg -specific current, is activated 2+ by C a (Preston, 1990). This novel current, the first ever reported in any organism, has a selectivity of 2+ 2+ 2+ 2+ 2+ 2 + Mg > Mn = Co > Sr = B a ^> C a , and is affected in some of the calmodulin mutants (R. R. Preston, unpublished results). Hyperpolarizations under voltage clamp also reveal 2+ at least five currents. (1) 7 C a ()h is an unusual C a cur2+ 2+ rent. Unlike other C a currents, B a not only cannot carry but blocks this current (Preston et al, 1992a). The current is also blocked by amiloride and shows 2+ a C a -dependent inactivation (Preston et al, 1992b). + (2) 7 K ( h/ ) a hyperpolarization-activated K current, is blockable by quinidine (Satow and Kung, 1977; Oertel a + et al, 1978; Preston et al, 1990a). (3) 7K(Ca,h)/ K current, is activated after J Ca(n)- This current is kinetically and pharmacologically distinct from 7 K ca,d) ( (Preston et al, 1990a). Some calmodulin mutations of the pantophobiac type reduce this current, whereas rst causes an early activation of it (Preston et al, a n 1990b, c). (4) I N a ( C) ais induced by / C (ah ) / d is believed 4 to flow through the same Na " channel activated by 2+ + C a from either 7 C a, since the N a currents at either polarization are affected by the fast-2 type of calmodulin mutant in an identical manner (Preston et al, 1991). a so 2+ (5) JMg(Ca) l rnost likely flows through the M g 2+ channel activated by 7Ca(d)/ since the M g currents observed at opposite polarizations have the same ion selectivity and blockage pattern (Preston, 1990). When Paramecium strikes an obstacle in its swim path, it backs up. Mechanical impacts of the anterior end of electrode-pinned cells elicit a depolarization prior to the action potential. This receptor potential 2+ was found to be based on C a (Eckert et al, 1972). Although nearly all divalent cations were able to carry t ne Jca(mech)/ corresponding receptor current under 2+ voltage clamp, C a presumably is the natural carrier based on the distributions of ions (Ogura and Machemer, 1980; Satow et al, 1983). Touching the poste-rior of a Paramecium causes it to spurt forward. This mechanical stimulation induces a hyperpolari+ zation by means of a K efflux, 7K(mech) (Naitoh and Eckert, 1973; Machemer, 1976; Ogura and Machemer, 1980).

450

BORIS

MARTIN

When a fraction of Paramecium ciliary membrane was incorporated into planar lipid bilayers, activities of two types of divalent-cation-passing channel were observed and reported (Ehrlich et al., 1984): (1) a voltage-independent 29-pS channel through which 2+ 2+ 2+ M g is slightly more permeant than B a and C a that might be equivalent to the anterior mechanoreceptor channel and (2) a voltage-dependent 1.6-pS 2+ 2+ 2 + channel that favors B a and C a greatly over M g , which might be the microscopic equivalent of / C a( d ) The application of patch clamp to surface blisters or detached cilia from Paramecium (Fig. 1) allowed recording of activities from many different types of ion + channel including at least four types of K channel, a 2+ + C a channel, a N a channel, a Cl~ channel ( J C 1) , and two cation-nonspecific channels (7 M+ ), one of which can be activated by ATP ( / M + (ATP)) (Martinac et al., 1988; Y. Saimi, B. Martinac, and R. R. Preston, unpublished observations). The activities of two channels 2 + have been documented more extensively. (1) A C a + dependent K channel of 72-pS conductance, that is more active on hyper- than on depolarization (Saimi and Martinac, 1989), appears to be the microscopic equivalent of / K(Ca,h)- Limited proteolytic digestion acti2+ vates this channel and makes it independent of C a 2 + and voltage (Kubalski et al, 1989). (2) A 19-pS C a + 2+ dependent Na channel, which depends on Ca -calmodulin for its activation (Saimi and Ling, 1990), is apparently the channel responsible for the macroscopic / N a c( a ) described earlier. For details on these two types of channels, see Preston et al. (1991) and Chapter 28. Although space limitations restrict our discussion to work on Paramecium, several ciliated protists have also been investigated in terms of their ion currents, including studies with two-electrode voltage clamp on Stylonychia (Machemer and Deitmer, 1987), Fabrea (Kubalski, 1987), and Stentor (Wood, 1982). Also studies of cation channels have been done by incorporating Tetrahymena ciliary membrane into planar lipid bilayers (Oosawa and Sokabe, 1985; Fujiwara et al., 1988).

III.

Dictyostelium

Dictyostelium discoideum is a slime mold that grows as individual amebas. On starvation, the amebas aggregate to form a multicellular fruiting body using cAMP as an intercellular signal. Aggregating amebas, alternating in time and in location with respect to neighbors in a swarm, release and respond to cAMP (Gerisch, 1987). Among other effects, binding of 2+ cAMP appears to stimulate a C a uptake (Bumann + et al., 1984) and a K release (Aeckerle et al, 1985).

AC

ET AL.

Mueller et al. (1986) first reported a patch-clamp study of Dictyostelium cells in which they found a + 9-pS channel that probably passes K . More recently, Mueller and Härtung (1990) reexamined the membranes of amebic cells after induction of differentiation by nutrient removal. In the cell-attached configuration, these researchers consistently observed the activities of three types of unit conductances: DI, an + 11-pS K conductance activated on depolarization; + Dil, a 6-pS K conductance activated on depolariza2+ tion; and HI, a 3-pS Ca -passing conductance activated on hyperpolarization. Based on probability of 2 encounter, channel densities appear low at —0.1 /μτη 2 for DI and HI and ~ 0 . 0 2 / μ η ι for DIL Like most channels in other microbes, the physiological roles of these channels have yet to be clarified.

IV. Yeasts a n d O t h e r F u n g i Because of its large cell size, Neurospora crassa, a bread mold, was the first fungus studied extensively using electrophysiological methods to investigate its membrane potential and how this potential is regulated (Slayman, 1977). A.

Plasma

Membranes

Two species of yeast have been used extensively in contemporary research: the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. These organisms can readily be cultured 2 + and manipulated in the laboratory. As expected, C a , + + 2 + K , Na , M g , and several trace metals are required 2+ for growth. Ca , in particular, is needed for cell-cycle progression (Iida et ai, 1990). In S. cerevisiae, at least two CDC proteins, products of cell-division cycle genes, have the EF-hand structures indicative of their 2+ being Ca -binding proteins (Baum et ai, 1986; Miyamoto et al., 1987). The plasma membrane of S. cerevisiae can form a gigaseal with pipettes after enzymatic removal of the cell wall (Fig. 1). Patch-clamp investigation showed activities of at least two types of ion channels. (1) A 20-pS channel is activated on depolarization above a threshold of - 1 0 to 0 mV and is not activated by hyperpolarization. This channel is highly specific for + + + K over N a and is blocked by the usual Κ -channel 2+ blockers tetraethylammonium and B a (Gustinetal., 1986). Surprisingly, Ramirez et al. (1989) assigned an + electrical activity to a K channel that appears at > + 1 0 0 mV or < - 1 0 0 mV in a wild-type. In a mutant defective in the structural gene for the plasma mem+ brane Η - A T P a s e , however, these investigators found this activity to appear at lower voltages in the

451

29. Microbial Channels presence of > 5 0 m M ATP. The mutation appears to + make this K channel sensitive to cytoplasmic ATP. Researchers suggested a tight physical coupling between the ATPase and the channel. (2) Stretch forces exerted on the membrane activate a 36-pS channel that is blockable by 10 μΜ gadolinium applied to the cytoplasmic side. This channel passes a variety of ions 2 + including C a , but prefers cations over anions. This channel also shows an adaptation to a sustained stretch force. Interestingly, this adaptation depends on voltage. The adaptation takes place when the force is applied when the membrane is polarized (cytoplasmic negative); a force applied during depolarization (positive) activates these channels but does not inactivate them. In whole-cell recording, previously inactivated channels can apparently recover during depolarization and force application. Experiments with whole spheroplasts of different sizes showed that the channel-activating force is a stretch along the plane of the membrane and not a pressure perpendicular to it (Gustin et al., 1988). Although this stretch force is most conveniently applied by suction to the pipette on the cell, positive pressure or osmotic forces applied by changing bath osmolality also activate these channels recorded in the whole-cell mode (X. L. Zhou, M. C. Güstin, and B. Martinac, unpublished observation). A failure to elicit the whole-cell macroscopic mechanosensitive current that was anticipated by microscopic currents seen in patches excised from snail neurons, in particular, cautioned against the possible artifactual origin of mechanically induced activities in general (Morris and Horn, 1991). The expected macroscopic currents have been recorded from S. cerevisiae spheroplasts (Gustin et al., 1988). As reported by Güstin, and to the relief of Morris and Horn (Gustin et al., 1991), not only can such macroscopic currents be recorded but they saturate at higher stretch forces, as expected of a finite number of open channels in a yeast cell and not expected of breakdown and leakage. Macroscopic currents from "whole spheroplasts" or "whole bleb" have also been observed in two other fungal preparations. Although the function^) of the mechanosensitive channels is not yet known, they are likely to play a role in osmoregulation, since yeasts show clear physiological responses to osmotic stress (Blomberg and Adler, 1989). All unicellular fungi are called yeasts. However, unicellularity is not a profound trait; fission yeasts and budding yeasts were apparently separated very early in evolution. Among the many differences are cell shape and wall compositions. Unlike the result for the budding yeast, treatment of the fission yeast S. pombe with zymolyase causes "protoplast protuberances" to appear at the pointed end of the cell (Kobori et al., 1989). These blebs can readily be studied with

a patch clamp. The more thoroughly documented ion channel from these blebs is a mechanosensitive channel (Zhou and Kung, 1992). Currents through one or two such channels, and ensemble currents of tens of units, have been recorded from excised patches and whole blebs, respectively. These mechanosensitive channels can be activated by slight pressure applied to whole blebs and can be blocked by gadolinium, like those in S. cerevisiae. Unlike the S. cerevisiae channels, however, the S. pombe channels are more conductive (180 pS) and more selective ( P K +/ P C 1_ = 3.6), and are poorly activated but strongly inactivated on membrane depolarization during force application. Reports have also been made of activities of other ion channels in the S. pombe plasma membrane. Larsson et al. (1992) noted the activity of a voltage-gated cation channel of four conductance levels; Zhou and Kung (1992) intimated three types of voltage-activated channel in addition to the mechanosensitive one in this membrane. More detailed reports are anticipated. The bean-rust fungus Uromyces appendiculatus is a curious organism of agricultural import. To successfully infect its host, a germ tube emerging from the spore must reach a stoma on the leaf surface. The growth of the germlings is apparently guided to the stomata by the topography of the bean-leaf surface. Differentiation of the germlings into infectious structures is triggered by the topography of the stomatal lips (Hoch et al, 1987). Treating germinated spores with Novozyme® converts them into protoplasts suitable for patch-clamp studies. Whereas voltage activates channels of smaller conductances, suction applied to the patch activates a 600-pS channel. This channel excludes Cl~ but is not very selective among + 2+ cations. Its permeability to K and to C a are nearly the same. Activities can be seen in excised patches as well as in whole protoplasts. The maxima of macroscopic currents indicate about 100 channels in a 4-μ,ιτι diameter protoplast, or about 2 channels per 2 μπ\ (Zhou et al., 1991). This current could transduce the membrane stress induced by the leaf topography 2 + into an influx of C a , which could then act as a second messenger for subsequent differentiation of the infectious structures. However, causal relationships have yet to be established. Note that gadolinium blocks this channel and also prevents germling growth and differentiation. B. Internal

Membranes

As diagrammed in Fig. 1, hypo-osmotic rupturing of the plasma membrane of S. cerevisiae releases a giant mitochondrion and a vacuole (Saimi et al, 1992). Both objects can be investigated with patch-clamp techniques.

452

BORIS MARTINAC

Although first discovered in mitochondria of Paramecium (Schein et al, 1976b), the outer membranes of all mitochondria tested, including those of yeast, are equipped with voltage-dependent anion-selective channels (VDACs). Through these channels, nonprotein molecules traverse the mitochondrial outer membrane. Direct examination of the mitochondria, exposed as shown in Fig. 1, revealed activities of VDAC (Saimi et al, 1992). Wunder and Colombini (1991) studied thoroughly the VDAC of Neurospora (a bread mold) reconstituted in liposomes with patch-clamp techniques. Most of the experimentation on VDACs, however, was performed with planar lipid bilayers. VDACs are highly conductive. The open-state conductance is 14 nS in 4 M KCl and is even higher in higher KCl concentrations. Open VDAC is mildly ° f 1-8. VDAC also anion selective, with a Pq\-/PK+ shows several lower-conductance "closed" states of different ion selectivity. The channel tends to open when no voltage is imposed, but closes when the membrane is polarized in either direction. The VDAC of S. cerevisiae is a 30-kDa protein consisting of 283 amino acid residues (Mihara and Sato, 1985). The sequence contains no hydrophobic region long enough to span the bilayer as an a helix, except an N-terminal amphipathic α-helical mitochondrial target sequence. However, the VDAC sequence contains 12 stretches of alternating hydrophobic and hydrophilic residues, which could form a β sheet with the former protruding on one side and the latter on the other (Forte et al, 1987). The current model of VDAC is a barrel of a single such β sheet. This model explains why so little protein can enclose such a large pore. Extensive sitedirected mutagenesis showed that charge-reversal mutations in sites distributed throughout the length of the molecule can alter ion selectivity. These sites apparently form the filter lining of the pore (BlachlyDyson et al, 1990). Voltage-induced closure to lowerconductance states is modeled as having some of the β strands slip out of the barrel, causing it to become narrower. At some sites, charge reversals alter the ion selectivity of the open state but not of the closed states (Peng et al., 1992). These sites may be on β strands that are extruded in the closed states. Zimmerberg and Parsegian (1986) found a large decrease in the volume of water within VDAC when it closes. This observation is also consistent with the stave-removal model, and not with a local-constriction model for closure (see also Chapters 34 and 35). Figure 1 also shows the release of a vacuole from a yeast protoplast after osmotic shock. Patch-clamp examinations of such vacuoles, in vacuole-side-out excised patches, have been carried out. Several types of conductance were noted, including two studies dis-

ET AL.

cussed in more detail (Saimi et al, 1992). For example, Minorsky et al. (1989) noted activities of a cation conductance that is 140 pS (at cytoplasmic-side positive voltages) and 98 pS (vacuole positive), with subconductance states and a slow kinetics. Berti and Slayman (1990) made a more thorough study of an ~120-pS channel and showed that it activates at a range of cytoplasmic-side negative voltages. Channel opening 2+ is not affected much by cytoplasmic C a until it exceeds 1 m M . Interestingly, however, reducing agents cause the channel to become activated at micromolar 2 + levels of cytoplasmic C a . C. Killer

Toxin

Some yeast strains produce toxins that are capable of killing cells of sensitive strains. The K l killer strains of S. cerevisiae harbor an M l virus with a doublestranded RNA genome. The K l killer toxin, the product of a viral gene, forms a 118-pS cation channel when incorporated into artificial liposomes (Martinac et al., 1990a; Fig. 1). This channel gates independently + of voltage and is capable of passing K . It does not + + discriminate between K and Na , but prefers monovalent to divalent cations. These findings are consis+ tent with the notion that K depletion of the sensitive cells during intoxication is responsible for the lethal effect.

V. Escherichia

coli

Escherichia coli, like other gram-negative bacteria, has two membranes separated by periplasm. Peptidylglycan cell wall in the periplasmic space provides rigidity for the cell. The wall and two membranes are collectively referred to as the cell envelope. The outer membrane is a very specialized structure because the outer monolayer is composed of lipopolysaccharide rather than the usual phospholipids. At least 14 proteins are usually found in this membrane, among which are porins (see subsequent discussion). The inner membrane is the true cytoplasmic membrane, which houses the machinery for electron transport, nutrient uptake, chemoreception, and so on. 5 Some 10 porins constitute a major portion of the outer membrane of an E. coli cell. Although other types of inducible porins for special solutes exist, this review covers only the group of major porins comprising OmpC, OmpF, and PhoE, all of which show more than 70% homology in their amino acid sequences (Jap and Walian, 1990; Jeanteur et al, 1991). Although these porins form trimers, each ~30-kDa monomer is an individual aqueous channel that can pass hydro-

29. Microbial Channels philic solutes up to 600 daltons in size. The structures of OmpF and PhoE in the open state have been solved by X-ray crystallography (Cowan et al., 1992). Both appear to be trimeric. Each monomeric subunit consists of a 16-stranded antiparallel β barrel enclosing a pore. A loop between β strands 6 and 7 folds into the pore, constricting the pore at about half the height of the barrel to 7 x 11 À in an elliptical cross section. This constriction apparently defines the conductance, since selected OmpC mutations that increase the solute-exclusion size limit are substitutions of sites in this loop by residues with smaller side chains (Misra and Benson, 1988). Lakey et al. (1991) showed that this constriction apparently also defines selectivity, since a lysine-to-glycine difference at a strategic position of this loop correlates well with the difference in the slight anion preference of PhoE and the slight cation preference of OmpF. Extensive biochemical studies led to the view that porins are responsible for the molecular sieve nature of the outer membrane (Benz, 1988; Nikaido, 1992) and that porins are static pores. However, some studies in planar bilayers showed that porins can be closed by strong membrane polarizations (Schindler and Rosenbusch, 1978; Morgan et al, 1990). Berrier et al. (1992) used a patch clamp to examine OmpF and OmpC porins purified from salt-extract reconstituted in liposomes. These porins could be closed by strong voltages in steps of 200 pS, but also showed millisecond flickers in steps of 6 0 70 pS at lower voltages. Berrier et al. (1992) interpreted these results to be the behaviors of the trimers and the monomers, respectively. Using a method modified from that of Criado and Keller (1987), Delcour et al. (1989a) incorporated E. coli membrane fractions into asolectin liposomes and found three types of channels. One type tends to be open most of the time, but frequently and briefly exists in the closed state. Gating of several units between states appears to be cooperative and is encouraged by depolarization. Strong depolarization irreversibly closes the channels, however (Delcour et al, 1989b). As mentioned earlier, Misra and Benson (1988) generated OmpC mutants that could survive on a substrate larger than the solute-exclusion limit of wild-type OmpC. Lakey et al. (1991) showed that this mutant has a larger pore and a greater voltage sensitivity using planar lipid bilayers. Using this mutant, Delcour et al. (1991) identified the activity, observed with liposome patches, to be that of OmpC. These investigators showed that the mutant channel has a 9-30% increase in unit conductance, as expected of this mutant, which is known to have a larger solute-exclusion limit. However, the most prominent change is that the mutant channel shows a voltage-dependent closure, mostly in groups of three

453

rather than in groups of two, as seen in the channels of the parent strain. The differences in porin kinetics observed by Delcour et al. (1991) and Berrier et al. (1992) may be explained by the use of incorporation of membrane fragments in the former study and the use of purified porins in the latter study. However, why OmpC porins appear to be more active in patchclamp experiments than in planar bilayer experiments is not clear. Ascribing a physiological function to voltage-gating of porins is also challenging. However, a Donnan potential occurs across the outer membrane. If periplasmic charges are organized and are intimately associated with the inner monolayer of the outer membrane, the potential across this membrane, as experienced by porins, may be larger than can be estimated (Stock et al., 1977). Major contributors to these charges are the membrane-derived oligosaccharides (MDOs) in the periplasm. Interestingly, Delcour et al. (1992) showed that MDO not only reduces the OmpC currents but also promotes their cooperative closures. Osmolarity regulates the synthesis of both porins and M D O . Low osmolarities in the culture medium induce synthesis of MDO, the concentration of which often rises up to 20 m M (Sen et al, 1988). MDOs as fixed negative charges in the periplasm should attract positive charges to the periplasmic space. These molecules may adjust the ionic strength and, therefore, the osmolarity of the periplasm, which then acts as a buffer for the cytoplasmic (inner) membrane against low osmolarity in the external medium. However, changing MDO concentrations in the periplasm does not affect the permeability of cephaloridine as measured by a substrate-conversion method (Sen et al., 1988). Low medium osmolarities also favor higher OmpF/OmpC ratios in the outer membrane of strain K-12. The elaborate molecular mechanism that regulates the OmpF/OmpC ratio has been studied by genetic dissection (Igo et al, 1990). The adaptive value of this regulation is not clear, however, since K-12 mutants lacking OmpF or OmpC and the closely related wild-type strain Β lacking OmpC all grow well in the laboratory, regardless of culture osmolarity (see Csonka and Hanson, 1991). The surfaces of giant spheroplasts or giant cells generated by the method shown in Fig. 1 or by other methods (Buechner et al., 1990) can form gigaOhm seals with patch-clamp pipettes (Martinac et al, 1987). Gigaseal formation is itself surprising, given the conventional view of the outer membrane as a static sieve. Either the outer membrane is dynamic and most porins are closed in vivo, or the patch-clamp manipulation induces porin closure. In any event, patch-clamp studies revealed the activity of a mechanosensitive

454

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MARTINAC

channel in such a preparation (Martinac et al, 1987). Mild suction or pressure greatly increased the open probability of this channel in a manner predictable by a Boltzmann distribution in which the applied mechanical energy partitions the channel between its open and closed conformations. An osmotic change in the solution bathing an inside-out patch can also activate this channel (Martinac et al., 1992). Gadolinium ion at submillimolar concentrations blocks this channel (Martinac et ai, 1991). Membrane depolarization favors the open state. This channel has a conductance of about 1 nS in 200 m M KCl. The channel visits substates of lower conductances. The dwell time in these substates is much longer in a mutant lacking the major lipoprotein (Kubalski et al, 1992a). The channel favors the passage of anions slightly over cations, and its kinetic behavior is affected by the ionic species (Martinac et al., 1987). Higher levels of suction applied to spheroplast patches through the pipette often activate a second channel that has an approximately 3-nS conductance and much faster open/close kinetics than the 1-nS channel (Sukharev et al., 1993,1994). Szabo et al. (1990) also reported mechanosensitive currents from E. coli spheroplasts. These researchers observed a much larger number of conductances in a wild-type than the two studied by Sukharev et al. (unpublished data). Differences in strains and procedures may account for some of the differences. Escherichia coli mechanosensitive channels in membrane fragments have been reconstituted into asolectin liposomes and were found to retain their functions observed in spheroplasts, including their ability to be activated by mild suction (Delcour et al., 1989a). The ability to reconstitute the channel activities in vitro (Sukharev et al., 1993) allowed enrichment and identification of channel proteins by following the activities through fractionation. Beginning with total £. coli membranes, two different series of chromatography enriched the same protein of about 17,000 m.w. corresponding to the 3-nS activity. N-terminal sequence of this protein led to the cloning of its gene, mscL (mechano-sensitive channel of very Zarge conductance). Insertional disruption of mscL (knockout) removed the channel activity. Expressing a plasmid-born mscL in the mscL-knockout chromosomal background restored the activity. Furthermore, the 17-kD protein was also expressed in a cell-free reticulocyte lysate with mscL being the only template and the expressed material containing the channel activity. The open reading frame of mscL corresponds to a unique protein of 136 amino-acid residues. The N-terminal f of the molecule is highly hydrophobic (Sukharev et al, 1994). Unlike channels gated by ligands or voltage, little is known concerning mechanosensitive channels in terms of molecular structure.

ET AL.

mscL is the first such channel to be cloned and sequenced. Deeper understanding of mechanosensitivity will be reached by dissecting mscL as a model molecule. Because this channel can readily be incorporated in vitro and function in foreign lipids, it has a very large conductance and therefore a favorable signal-to-noise ratio. Also, it resides in the genetically most amenable biological system, that of E. coli. Although a biochemical and molecular biological approach to studying these channels will be crucial to our understanding in the future, a biophysical approach has been taken to determine how the force is transduced to the channel protein. Cell membranes are asymmetrical; the inner monolayers have more phospholipids with negatively charged head groups than the outer monolayers (Rothman and Lenard, 1977). The outer membrane of E. coli is even more asymmetrical, since its outer monolayer is of an unusual composition. An amphipathic molecule is expected to partition differentially into the two monolayers because of this asymmetry. The uneven insertion of amphipaths leads to local membrane buckling, which is readily visible in blood cells, as predicted by the "bilayer couple hypothesis" (Sheetz and Singer, 1974,1976). Such a perturbation is expected to create a mechanical stress force in the membrane. Martinac et al. (1990b) showed that a variety of amphipaths activate the 1-nS mechanosensitive channel of E. coli. Further, channel activations by different amphipaths correspond in time course and effectiveness to their known solubilities into lipid. In animal cells, the gating force is believed to be transmitted to the mechanosensitive channels through the cytoskeleton (Sachs, 1988). The amphipath experiment and the reconstitution experiment in E. coli indicate that the channel can respond to stress forces in the lipid bilayer instead. Sukharev et al. (1993,1994) showed that the E. colt mechanosensitive channels, solubilized in a detergent and reconstituted into liposomes of foreign lipids, nonetheless retain their mechanosensitive function. Thus, at least these types of channels are gated by tension transduced exclusively via the lipid bilayer. In E. coli, the outer membrane is anchored to the peptidylglycan (cell wall) that provides rigidity. Digestion of this peptidylglycan with lysozyme activates rather than inactivates the channel (Buechner et ai, 1990; Martinac et al., 1992). Peptidylglycan, the bacterial equivalent of the cytoskeleton, normally restrains the outer membrane, attenuating rather than transmitting the mechanical forces in the membrane (Martinac, 1992). Membrane deformation can also be induced by mutations. The most abundant protein in the outer membrane is the 7.2-kDa lipoprotein that is embedded in the inner monolayer and does not protrude into the

29. Microbial Channels outer monolayer. This lipoprotein is also a major covalent link between the outer membrane and the peptidylglycan. Kubalski et al. (1993) showed that the outer membrane of a mutant lacking this major lipoprotein is much less able to transmit the stretch force to the mechanosensitive channel. A bulky amphipath (lysolecithin), but not smaller amphipaths, can restore this ability. The forces that gating the channel in the bilayer that result from the relative but simultaneous expansion and compression of the two monolayers have been treated in a theoretical model by Markin and Martinac (1991). The location of the 1-nS mechanosensitive channel described in the preceding section is controversial. Martinac and co-workers (1987,1992; Buechner et al, 1990) argued for its outer membrane location on several grounds. Giant spheroplasts have an outer membrane as seen by electron microscopy and immunofluorescence microscopy. The patch-clamp pipette is not likely to be able to pass through the outer membrane and still seal onto the inner membrane. Digestion of excised inside-out patches with lysozyme added to the bath activates the channel. Were the channel in the inner membrane, the peptidylglycan would have been facing the pipette side, inaccessible to lysozyme. Also, mutational deletion of lipoproteins clearly located in the outer membrane strongly affects the behavior of these mechanosensitive channels, as described earlier (Kubalski etal., 1993). Finally, patchclamp experiments on protoplasts with only the cytoplasmic membrane revealed the activities of a new set of channels but not the activities of porins or the 1-nS mechanosensitive channel (Kubalski et al., 1992). Note that these arguments are based on observations made on patches from spheroplasts or protoplasts, preparations more closely resembling live cells than reconstituted liposomes. Even these investigators found mechanosensitive channel activities in asolectin liposomes incorporated with material from either the "inner membrane" or the "outer membrane" fractions, separated by sucrose gradient (Delcour et al., 1989a). A different group of investigators (Berrier etal., 1989) observed mechanosensitive channel activities in 50% of the patches from liposomes incorporated with inner membrane material, but only in 7% of the patches from those incorporated with outer membrane material, and concluded that the channel is localized to the inner membrane. Conclusions must be drawn cautiously because perfect separation of inner and outer membranes by gradient centrifugation cannot be achieved (Osborn et al., 1972) and a direct comparison of different material in liposomes assumes the same efficiency of incorporation and the same degree of channel inactivation in the procedures. Berrier et al. (1989) found the mechanosensitive channel(s) to

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be of different unit conductances, from 140 to 950 pS. Events of even higher conductance were observed, although rarely. The differences between the observations by Berrier et al. (1989) and Delcour et al. (1989a), both made on reconstituted liposomes, have not been resolved. As early as the 1950s, investigators observed that hypotonic shock caused £. coli to jettison its smaller solutes and ions but retain its macromolecules (Britten and McClure, 1962). The pathway for this behavior has not been identified, but it clearly has an exclusion limit. Building on the idea that the mechanosensitive channels observed are in the inner membrane, Berrier et al. (1992) showed that gadolinium ion blocks the channel and also blocks the loss of ATP, glutamate, + and lactose and slows down the loss of K from osmotically down-shocked cells. These researchers concluded that this channel is the pathway for the controlled efflux of solutes on hypo-osmotic shock. Although this idea is very attractive, caution is required since gadolinium blocks most if not all mechanosensitive channels. Based on the methods of Birdsell and Cota-Robles (1967), Kubalski et al. (1992) succeeded in preparing E. coli protoplasts suitable for patch-clamp experimentation. Several types of ion channels, each less than 100 pS in conductance, have consistently been observed. In addition, these investigators have also observed a 600-pS mechanosensitive channel that has kinetics that are completely different from those of the 1-nS or 3-nS channels observed on the outer membrane of E. coli (Kubalski, B. Martinac, and Kung, unpublished observations). Gram-positive bacteria have only one membrane, the cytoplasmic membrane. Note that mechanosensitive channels have also been recorded from the membranes of two such bacteria: Streptococcus faecalis (Zoratti and Petronilli, 1988) and Bacillus subtilis (Zoratti et ai, 1990; Martinac et al, 1992). Using gradient-fractionated inverted vesicles of E. coli plasma membrane fused to planar lipid bilayers, Simon et al. (1989) detected a 115-pS channel at 45 m M potassium glutamate. This channel prefers anions over cations and was more active on negative voltages. Researchers believe it is a channel that conducts protein to the periplasm.

VI. Conclusion The application of a variety of techniques, especially patch clamp, has clearly shown that microbes, like other organisms, have ion channels. To date, microbial ion channels are largely only known by their activities. Except for VDACs, porins, and Shaker-like

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channel genes of Paramecium (T. Jegla and L. Salkoff, personal communication), the proteins and genes that correspond to these electrical activities have yet to be identified. As stated at the beginning of this review, microbes are vastly more diverse than animals and plants. That different types of channels are encountered in different microbial membranes is, therefore, not surprising. + Interestingly, voltage-gated Κ -specific channels seem to have evolved in all branches of eukaryotic organisms, including plants, animals, protists, slime 2+ molds, and several fungi. Voltage-gated Ca -specific channels are found in Paramecium and Dictyostelium, + but not in yeasts. Voltage-gated N a channels have + not been encountered in microbes to date. Such Na channels might represent late evolutionary modifica2+ tion of C a channels, to exploit only the electrical and not the ionic effects. Also no reports have been made on microbial channels directly gated by external ligands. This deficiency may not reflect an absence of such channels from microbes as much as the difficulty in knowing which of the hundreds of possible ligands should be presented to the microbial surfaces. Gating by internal second messengers is exemplified by four 2+ types of Ca -gated channels in Paramecium. Analyses 2+ show clearly that these channels are Ca -calmodulin gated. This method of gating is apparently not limited to protists, since evidence suggests that this mechanism is also employed by higher animals (see Chapter 28). Determining whether other current types first 2+ described in microbes, such as Mg -specific current 2+ and hypolarization-activated C a current, also have equivalents in higher forms would be interesting. Channel gating by stretch force in the lipid bilayer appears to be an ancient mechanism. Mechanosensitive channels are found in Paramecium, yeasts, E. coli, and other bacteria. This fact may also reflect the need common to all microbes to deal with osmotic stress. The discovery of ion channels in the cytoplasmic membranes of bacteria is surprising to many bacteriologists. Intuitively, the cytoplasmic membrane sustaining a proton motive force should not be leaky. This intuition leads to a claim that bacterial cells, unlike eukaryotic cells, do not have ion channels except in the outer membranes (Maloney, 1987). This claim originated from the absence of reports of channels in the inner membrane. However, until recently, no proper technique has been available by which to evaluate this membrane properly, electrophysiologically. The use of patch clamp, in combination with other methods, should provide a better understanding of bacterial electrophysiology. Although E. coli clearly has ion channels in its outer and inner membranes, as reviewed here, the relationship of these channels to eukaryotic channels has yet to be established.

ET AL.

Many basic mechanisms, such as how protein conformation can be governed by a stretch force, remain to be elucidated. How microbial channels function in the lives of microbes is also largely unknown. Clearly, future studies on the molecular biology and molecular physiology of microbial channels hold promise that is likely to be fulfilled, since the microbes reviewed in this chapter are amenable to genetic and molecular manipulations. Acknowledgments We would like to thank Yoshiro Saimi, who introduced the patch-clamp technology into our laboratory, making it possible for us to examine single-channel activities of Paramecium, yeasts, and E. coli. We would also like to thank Robin R. Preston for constructive criticism of this paper. This work was supported by National Institutes of Health Grants GM22714 and GM36186 and a grant from the Lucille P. Markey Charitable Trust.

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