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Structure and Selectivity of Porin Channels
CURRENT TOPICS IN MEMBRANES A N D TRANSPORl. VOLUME 21
Structure and Selectivity of Porin Channels R . BENZ Dcpartmcnt of Biology Univcrsity of Konstanz Konstanz, Fcdcral Republic of Gcrmuny
I . Introduction. ...... 11. Reconstitutio rins in .............. A. Addition of Porin to the Aqueous Phase.. . . . . B. Membrane Formation from C. Fusion of Reconstituted Ve 111. Single-Channel Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Ionic Selectivity of Porin Channels . . . . . . lective Channel V. Properties of the .............. Outer Membrane VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
199
200 200 203 204 205 207 209 213 211
INTRODUCTION
The cell wall of gram-negative bacteria consists of three different layers, the inner membrane, the peptidoglycan layer, and the outer membrane (Di Rienzo et al., 1978; Nikaido, 1979a).The inner membrane acts as a real permeability barrier and contains, in addition to the respiration chain, a large number of different transport systems for substrates, whereas the peptidoglycan layer prevents lysis of the bacterium. The outer membrane seems to act a5 a molecular filter for hydrophilic wbstances and as a barrier for hydrophobic molecules (Nikaido, 1979b; Nikaido and Nakae, 1979). The sieving property of the outer membrane of gram-negative bacteria resides in a defined exclusion limit for hydrophilic solutes. The permeability of the outer membrane I S associated with the presence in the outer bacterial membrane of a class of major proteins called matrix proteins (Rosenbusch, 1974) or porins (Nakae, 1975, 1976). 199 Copyright CJ 1984 by Academic P r e s . Inc All right\ ot reproduction in dny form reserved
ISBN n-12-153121-2
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Porins have been isolated from the outer membrane of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa (Hancock et al. , 1979, 1982; Nakae, 1976; Nakae and Ishii, 1978). They form trimers in dodecyl sulfate that are usually stable. Experiments on reconstituted vesicles in the presence of such porin trimers from enteric bacteria have shown that the maximum molecular weight for permeable substances in the case of E. coli and S. typhimurium is between 600 and 700, whereas molecules with molecular weights up to 6000 can penetrate vesicles reconstituted in the presence of porin F from P. aeruginosa (Hancock et al., 1979; Nakae, 1976; Nakae and Ishii, 1978). Experiments on reconstituted vesicles provide valuable information on the presence and the size of the pores formed by a porin. More detailed information about the pore interior and pore selectivity can be obtained from experiments on lipid bilayer membranes. The reconstitution of the porins into lipid bilayers and the properties of the porin channels from E . coli, S. typhimurium, and P. aeruginosa in lipid bilayer membrane are described in this article. In lipid bilayer membranes the porins of these gram-negative bacteria form, in general, large water-filled pores with a poor selectivity for ions. However, there are exceptions. Protein P from P . aeruginosa outer membrane forms highly anion-selective channels in lipid bilayer membranes (Benz et al., 1983; Hancock et al., 1982). II. RECONSTITUTION OF PORINS INTO LIPID BILAYER MEMBRANES A. Addition of Porin to the Aqueous Phase
Three different methods have been used successfully to reconstitute porins into lipid bilayer membranes. Firsi, detergent-solubilized porin is directly added to the aqueous phase bathing a membrane (Benz et al., 1978a); second, the lipid bilayer membrane is formed from reconstituted vesicles according to the Montal-Mueller method (Schindler and Rosenbusch, 1978); and third, the porin is inserted into the planar bilayer via fusion of the reconstituted vesicles with the membrane (Cohen et al., 1982). The simplest method consists of the addition of purified porin from a stock solution containing. 0.1% dodecyl sulfate or Triton X-100 to the aqueous phase bathing a black lipid bilayer membrane (Benz et af., 1978a). Figure I shows such an experiment. Porin from S. typhimuriurn strain SH 5551 (M, 40,000) was added in a final concentration of 100 ng/ml
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
10-8
201
3 0 10 20 30 LO t (rnin)
FIG.I . Specific membrane conductance as a function of time after addition of 100 nglml trimers of S. tvphinirrrirrm strain SH 5551 ( M ,40.000) to a black membrane from egg phosphatidylcholineln-decane (closed cir'cles, arrow). The open circles represent a control experiment in which only 10 pg/ml SUS was added to another membrane. I M KCI. 25°C.
to a black membrane from egg phosphatidylcholine/n-decane.After an initial lag of 4 minutes, presumably due to diffusion of the protein through unstirred layers, the conductance increased by about three orders of magnitude within about 30 minutes. Only a slight additional increase (as compared with the initial one) occurred after that time. It has to be stressed, however, that in the presence of all porins the membrane conductance increases continuously until membrane breakage. It is interesting to note that detergents had only a small influence, if any, on porin reconstitution. This was shown by using various detergents and detergent-free porin from osmotic shock solution (Benz et id., 1978a,b). Porin trimers dissociated into monomers were found to be inactive in lipid bilayer membranes and reconstituted vesicles (Nakae et a / ., 1979). Since in all the experiments with porins a steady conductance level could not be reached, the dependence of the conductance on various parameters was difficult to determine. A meaningful comparison was possible, however, on the basis pf experiments similar to those presented in Fig. 1, using the conductance value at a fixed time after addition of the protein. Figure 2 shows the influence of the membrane composition on the incorporation of the porins from S . typlzirnrrrium strain TA 1014 ( M , 38,000, 39,000, and 40,000) into the membranes. For membranes made from oxidized cholesterol or monoolein, the conductance is about two to
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Protein concentration (ng/rnl)
FIG.2. Specific membrane conductance A as a function of the concentration of trimers from the Salmonella strain TA 1014 ( M , 38,000, 39,000, and 40,000) in the aqueous phase; 1 M KCI; T = 25°C. Less than 5 pg/ml SDS was present in the aqueous solutions. The membranes were formed from different lipids dissolved in n-decane. The measurements were performed 20 minutes after blackening of the membranes. (0) Oxidized cholesterol; ( W monoolein; ( 0 )brain phosphatidylserine; (0) egg phosphatidylcholine; ( X ) egg phosphatidylethanolamine.
three orders of magnitude larger than for membranes made from phospholipids. A similar “lipid specificity” was also found for the porins from E . coli and P. aeruginosa (Benz et al., 1978a; Benz and Hancock, 1981). It is obvious that many conductive units were incorporated into lipid bilayer membranes in the experiments described above. This allows investigation of the current-voltage behavior of membranes containing a large number of porin trirners. Examples of current-voltage curves of two membranes made from oxidized cholesterolln-decane in the presence of porin trimers of 3. typhimurium strain HN 6017 ( M , 38,000) are given in Fig. 3. The observed current was a linear function of applied voltage up to at least 150 mV. Even for an application of 100 mV for more than 30 seconds no current decrease occurred, but sometimes a slight increase was found-presumably caused by the incorporation of the porin into the membranes, facilitated by the high electrical field. Linear current-voltage relationships were also found for porins from E . coli and P. aeruginosa (Benz et al., 1978a; Benz and Hancock, 1981; Hancock et al., 1982). The results strongly suggest that no voltage is required to initiate the single conductance unit, i.e., the pores are not voltage gated if the porin molecules are reconstituted by addition to the aqueous phase.
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
203
FIG.3. Current vs voltage characteristics of two membranes from oxidized cholesterol/ n-decane doped with I ng/ml porin ttimers from S. fypypAimitrium strain H N 6017 ( M , 38,000). The aqueous phase contained 1 M KCI and 50 ng/ml sodium dodecyl sulfate; 7 = 25°C.
B. Membrane Formation from Reconstituted Vesicles
Schindler and Rosenbusch (1978) used an alternative method for incorporation of porin molecules into lipid bilayer membranes. Vesicles reconstituted from lipids and porin are added to the aqueous phases on both sides of a thin Teflon foil having a small circular hole (100-200 pm in diameter) above the initial water level. Monolayers form on the surfaces of the two aqueous compartments. It is suggested by Schindler and Rosenbusch (1978) that these monolayers have the same composition as the vesicles. The water levels on both sides of the foil are now raised above the hole and a “folded” membrane is formed across the hole (Montal and Mueller, 1972). No pores are observed in the membrane immediately after formation at low membrane voltages. Membrane potentials around 100 mV are needed to induce the pores in steps larger than one single conductive unit (Schindler and Rosenbusch, 1978, 1981). Figure 4 shows such an experiment taken from Schindler and Rosenbusch (1978). The initial jumps indicate the formation of pore multimers in 0.1 M NaCl at 240 mV, whereas the subsequent decay is due to inactivation of the single conductive unit, caused by the high voltage. Schindler and Rosenbusch (1978) suggested the existence of three pores in a porin trimer. According to their view, all three pores should activate together but inactivate separately (see also Fig. 4). As a consequence of their observations, Schindler
i
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0
m
05 0
4 0‘
2
10
15
1 20
Time [minl
FIG.4. Initiation of conductance in a porin-containing membrane made from monolayers. The left side shows large current .jumps which appear at 240 m V membrane potential in 0. I M NaCI. Each large increment was followed by a decay in small steps (0.14 pS) as shown on the right side of the figure. (Taken with permission from Schindler and Rosenbusch, 1978.)
and Rosenbusch (1978) proposed a voltage control of the porin pores in the outer membranes of gram-negative bacteria. C. Fusion of Reconstituted Vesicles with Planar Lipid Bilayers
The third possibility of incorporation of porin molecules into planar lipid bilayer membranes is through fusion of reconstituted vesicles with planar bilayers. Miller and Racker (1976) and Miller et a1 (1976; and see Miller et ul., this volume) showed that cytochrome oxidase vesicles and vesicles from sarcoplasmic reticulum could be fused with planar bilayers in the presence of calcium ions. The calcium ions are most probably needed for the close apposition of the vesicles to the membranes. The yield of fusion could be drastically increased if an osmotic gradient hyperosmotic on the cis side (the side of the addition of the vesicles) were applied across the planar bilayer. The osmotic gradient results in a water flux toward the cis side. This water flux for unknown reasons accelerates the fusion process of the lipid vesicles with the planar bilayer. Recently, Cohen et al. (1982) used this method to incorporate porin molecules into planar bilayers. Upon addition of calcium ions to the cis side and establishing an osmotic gradient hyperosmotic at the cis side, the fusion process begins as indicated by a stepwise increase of the membrane current. The fusion process stops after the osmotic gradient is removed by addition of the osmotic active substance to the trans side. Furthermore, Cohen et ul. (1982) could show that water flux into the reconsti-
STRUCTURE AND SELECTIVITY
205
OF PORIN CHANNELS
tuted vesicles without any gradient across the planar bilayer also increases the yield of fusion. It is interesting to note that the fusion process resulted immediately in open pores in the lipid bilayer membranes and that no voltage-induced activation of pores was observed by Cohen et ul. (1982). Only a minor inactivation was found at high voltages prior to membrane breakage.
I II. SI NOLE-CHAN NEL AN ALYSl S The addition of small amounts (1-10 ng/ml) of porins to the aqueous phase bathing a membrane of small surface resulted in a stepwise increase of the membrane current at H given voltage. These current jumps were not observed when only the detergents dodecyl sulfate or Triton X-100 were added to the aqueous phase in the same concentration as the porin. Figure 5 shows an experiment using porin from E . coli. As can be seen from Fig. 5 all steps were directed upward, whereas downward steps are only rarely observed. Similar results are also found for the porins from S. typhirnuriurn and P . aeruginosu. The lifetime of all porin pores was at least I minute, as could be seen from records extending over long times. Only porin F pores observed in the presence of total outer membrane from P . ueruginosa (Hancock and Nikaido, 1978) had a much shorter lifetime, on the order of 50-100 msec, although the absolute level of the pore conduc-
0.5nS 25 pA
FIG.5. Stepwise increase of the membrane current in the presence of 0. I ng/ml porin from E. coli K12 added to the q u e o u s phase containing 0.1 M NaCl; T = 25°C. The membrane was formed from egg phosphatidylcholineln-decane;V , = SO mV. The record starts at the left end of the lower trace and continue5 in the upper trace.
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3 :0 ;1 P
0
0
li
0.1
1
L 02
03
OL
A (ns)
FIG.6. Histogram of the conductance fluctuations observed with membranes from egg phosphatidylcholineln-decanein the presence of porin from E . coli K12, 0.1 M NaCI; T = 25°C. n, the number of single steps; V , = 50 mV.
tance was not changed as compared with purified porin F (Benz and Hancock, 1981). Most of the conductance fluctuations obtained with various porins were not uniform in size but were distributed over similar ranges. A histogram of the conductance steps measured with porin from E . coli is shown in Fig. 6. The fluctuations for the E . coli porin varied about fourfold. Similar distributions were found for all porins. Only the recently discovered anion channel formed by porin P from P . aeruginosa gave a narrow distribution of the conductance fluctuations (Benz et al., 1983; Hancock et al., 1982). Single-channel measurements in the presence of the porin were performed with a variety of different salts and concentrations. From records similar to those given in Fig. 5 the average conductance increment A was obtained by measuring a sufficient number (at least 50) of individual events. For all porins described in this review (except porin P from P . aeruginosa; Benz et al., 1983) the average pore conductance A was a linear function of the specific conductance u of the aqueous phase, i.e., the ratio Alu varied only little in contrast to A, which varied by two orders of magnitude. This is also reflected in Fig. 7, where the average pore conductance A for the porin F of P . aeruginosa and a porin of E . coli are given as a function of u.The data points could be fitted with straight lines. The same is valid for the porins from S . typhiniurium, although the
207
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
001.
0 FIG. 7. Average pore conductance A of porin from E . coli K I? (open circles) and protein F from P . a ~ r u g i n o s a(closed circles) given as a function of the specific conductance of the corresponding aqueous salt solution; T = 25°C.
ratio Alu showed a much larger variation for E. c d i and S . typhimrrrirrm than for P . creri4g:ino.s~porin F. This can be explained by the larger diameter of the P . uerrrginosu pore. which is also consistent with the larger value of Alu (Fig. 7) and with a vesicle permeability assay using carbohydrates of different sizes (Hancock and Nikaido, 1978; Nakae, 1976). I t is interesting to note that even large organic cations and anions such as Tris', N(CH2CH3);, and HEPES- were able to pass through the porin pores with little or no interaction with the pore interior.
IV. IONIC SELECTIVITY OF PORlN CHANNELS Further information on the structure of the conductance pathway created by the different porins may be obtained by studying the ionic selectivity of the pores using zero-current potential measurements. Figure 8 shows the results of such experiments obtained for E. coli porin oligomers (which contained two different porins called OmpF and OmpC) in mcmbranes from oxidized cholesterolln-decane. The zero-current potential was found to be positive on the more dilute side of the membrane. This indicated that porin oligomers from E. coli form cation-selective pores in lipid bilayer membranes. From the measured V , and the concentration gradient c"Ic' across the membrane, the ratio P J P , of the permeabilities
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c”/c‘
FIG.8. Zero-current membrane potential V , as a function of the ratio of the salt concentrations on both sides of membranes containing porin from E . coli K12. The membranes were made from oxidized cholesterolln-decane; T = 25°C. V , was positive on the more dilute side of the membrane, c’ = lo-* M .The lines were drawn according to the GoldmanHodgkin-Katz equation with the specified values of the permeability ratio P,IP,. ( x ) KCI; (@) NaCI.
P , for cations and Pa for anions was calculated according to the GoldmanHodgkin-Katz equation: RT PCdr+ Pact V , = - In F Pccr t Pact’ where R , T , and F have their usual meanings. Table I shows the permeability ratios P J P , for the different porins with KCI as a salt in the aqueous phase. The observed slight selectivity for cations or anions may be explained by the presence of negative or positive charges, respectively, in or near the pores. The Pho E porin pore is induced in E . coli outer membrane under conditions of phosphate deprivation (Tommassen and Lugtenberg, 1980). The assumption that charged groups are responsible for the selectivity is supported by the pH dependence of the selectivity. The permeability ratio P J P , for the porin pore decreases in the presence of a NaCl gradient from 2.8 at pH 9 to 1.2 at pH 3 (Benz et al., 1979). Further support arises from selectivity changes of chemically modified porin. For example, porin OmpF from E . coli completely loses its cation
209
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
'TABLE 1
ZERO-CURRENT POTFNTIAI s V,,, FOR
THE
PRESENCE O F A IO-POI
D
DIFFERENT PORINSI N KCI GRADENr"
Porin
pH
V,,,( m V )
E . co/i OmpF/OmpC (la/lb) OmpF/OmpC (lailb) OmpF/OmpC (Ia/lb) PhoE (Ic)
h 3 9 h
27 t 3 -5 t 2 30 3 -25t5
S. rypliimitrirtm M, 40,000
h
24 t 2
3.2 t 0,s
20
2.4 2 0.4 10
P.
"
3.8 t 0.9 0.80 0. I 4.6 -t 0.9 0.29 t 0.08
*
trerllgitloscl
F P
6 h
'' 4
-57 + 4
V , is the electrical potential of the dilute side (lo-? M ) minus the potentilt1 of the concentrated side (10.' M ) . The membranes were formed from egg phosphatidylcholineln-decane or diphytanoylphosphatidylcholine/n-decane;T = 25°C. The ratios of the permeability P, (cation) and P, (anion) were calculated from the Goldman-Hodgkin-Katz equation. The nomenclature for the E . c d i porins was taken from Lugtenberg (1981).
selectivity after amidation, i.e., PJP,, = 1 (Tokunaga e t a / . , 1981; Benz c't a / . , 1984b). V.
PROPERTIES OF THE ANION-SELECTIVE CHANNEL FROM Pseudornonas oeruginosa OUTER MEMBRANE
The outer membrane of P. u~rrcginosrrobviously has very special sieving properties, which make this organism quite resistant to most antibiotics (Angus rt u/., 1981; Hancock rt d . , 1982). One of these sieving properties is the relatively short lifetime of the porin F channel. Presumably also present in the outer membrane is protein P ( M , 48,000), which is induced when P. creruginow is grown on media with phosphate limitations. Protein P forms anion-selective channels in lipid bilayer membranes. Figure 9 shows zero-current membrane potential measurements on dioleylphosphatidylchol/nemembranes in the presence of protein P and a KCI gradient. The zero-current membrane potential was negative on the more dilute side and could be fitted reasonably well to the Goldman-Hodgkin-Katz equation assuming either a permeability ratio PJP, = 0 (Nernst equation, full line) or PJP,, = lo-* (broken line). According to
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C'/d'
FIG. 9. Zero-current membrane potentials V , of diphytanoylphosphatidylcholine/n-decane membranes measured as function of a KCI gradient in the presence of protein P from P . ueruginosu; T = 25"C, V,,,was negative on the more dilute side of the membrane, c'' = lo-* M . The lines were drawn according to the Goldman-Hodgkin-Katz equation assuming P J P , = 10-' (broken line) or PJP., = 0 (solid line); T = 25"C, pH 6.
this result, chloride permeates through the porin P channel at least 100 times faster than does the potassium ion (Benz et ul., 1983). The current fluctuations of the porin P channel from P . ueruginosu outer membrane are found to be fairly homogeneous, in contrast to the steps in current observed with porins from Salmonella (Benz et al., 1980) or E . coli (Benz et al., 1978a). Figure 10 shows current fluctuations observed with protein P on a diphytanoylphosphatidylcholine membrane. There all current steps were directed upward, closing pores being only rarely observed. This indicates a long lifetime, as is found for the other porin pores. The lifetime of single steps was found to be independent of voltage, and linear current-voltage relationships were observed up to 200 m V for the protein P channels. The single-channel conductance of the porin P channel was found to be independent of the type of cation present in the aqueous phase. For a 100 mM chloride solution the single-channel conductance was 160 pS, irrespective of the size and nature of the cation. A much larger variation of the single-channel conductance A was obtained if the anions were varied. Figure 1I shows the dependence of the single-channel conductance A of
-
21 1
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
-
1
100 pA
2nS
FIG. 10. Single-channel record of a diphytanoylphosphatidylcholine/n-decane membrane in the presence of 10 ng/ml protein P from P . aerrcginnsci and 1 M KF in the aqueous phase. A voltage of 50 mV was applied through calomel electrodes with salt bridges; T = 25°C.
100 mM salt solutions on the size of the halides. The corresponding cation was in all cases potassium. The conductance appears to be highest for chloride and lowest for iodide. Besides the halides, a variety of other anions, such as nitrate and nitrite, also permeate through the porin P channel, whereas in the presence of salts with large organic anions, such as HEPES- (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid), no conductance fluctuations could be observed at all. The origin of the anion selectivity of the porin P pore from P . ueruginostr outer membrane is, therefore, presumably a selectivity filter with a diameter of -0.6 nm containing at least one positive charge. This structure accounts also for the concentration dependence of the single-channel conductance. Figure 12 shows the dependence of the single-channel conductance on the KCl concentration c in the aqueous phase. A linear conductance-concentration relationship was observed only with KCl concentrations as small as 1 and 10 mM. The single-channel conductance approached saturation for higher salt concentrations, and no further conductance increase occurred above 0.3 M KCI. The conductance concen-
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21 2
3m/
Radius (nml
FIG.11. Single-channel conductance of the porin P channel in diphytanoylphosphatidylcholineln-decane membranes as a function of the ion radius of the anion according to Pauling. 50 mV was applied to the membranes through calomel electrodes with salt bridges; T = 25°C.
Id'
1
FIG. 12. Single-channel conductance of the porin P channel from P . aeruginosa outer membrane as a function of the potassium chloride concentration in the aqueous phase. The membranes were formed from diphytanoylphosphatidylcholineln-decane. 50 mV was applied to the membranes; T = 25°C. The solid line was drawn according to Eq. (2) with the values of Amax= 280 pS, and K = 20 M-I.
STRUCTURE AND SELECTIVITY OF PORIN CHANNELS
213
tration curve A,((.) given in Fig. 12 could be fitted easily assuming single occupancy of the pore (i.e,, one binding site for anions in the pore), using the following equation [Lauger, 1973; see also Eq. (17) of Lauger, this volume]: &(c) = [cK/(I + ~ K ) ] h o , , , , , (2) with the binding constant K = 20 M-' and a maximum conductance of 280 pS (solid curve in Fig. 12). VI.
CONCLUSIONS
Porins isolated from the outer membrane of gram-negative bacteria are able to form ion-permeable pores in lipid bilayer membranes. Three different methods can be used for successful reconstitution of the porins into lipid bilayer membranes; the implications of the different methods are discussed in more detail below. Most investigations were performed by addition of the proteins to the aqueous phase. The insertion into the membranes is in this case presumably controlled by hydrophobic forces because the reconstitution rate shows no dependence on the ionic strength of the aqueous phase (Benz et al., 1978a, 1980). The reconstitution rate has been found to be strongly dependent on the type of lipid used for membrane formation. The conductance and the ionic selectivity of the single conductance unit, however, are independent of the nature of the lipid. We believe, therefore, that the reconstitution rate does not reflect a specific lipid-protein interaction. Moreover, it seems that the insertion of the trimers into the membranes is governed by a kinetic process, for example the replacement of 100 lipid molecules (each of area 0.5 nm?) by one porin trimer (area 50 nm2; Nikaido, 1979a), which may need considerable energy (Benz ct al., 1980). The data presented here support the assumption that large water-filled pores are formed by most, but not all, porins. Let us first consider the large pores. These pores are permeable to the large organic ions Tris+, N(C2Hs):, and HEPES- without detectable interaction with the pore interior. Furthermore, pore conductance shows no saturation with increasing salt concentration in the aqueous phase, and the single-channel conductance was a linear function of the specific conductance of all salt solutions. The current-voltage characteristic was ohmic-which, too, is expected for a wide unselective channel. Nevertheless there exist some differences in the single-channel conductances. The single-channel conductance of porin F from P . nevuginosa was considerably higher than conductances observed for porins from E. (di and S. fyphirnrrrirrm (Table 11). This indicates that the diameter of the porin F pore is considerably
214
R. BEN2 TABLE I1 COMPARISON OF THE PORESFORMED B Y PORINS OF GRAM-NEGATIVE BACTERIA“ Pore
E . coli OmpF (Ia) OmpC (Ib) PhoE (Ic) LamB S . typhimurium M , 38,000 M , 39,000 M , 40,000 P . aeruginosa F P (1
A (nS)
d (nm)
Cross section (nm’)
1.9 I .9 1.7 2.7
1.3 I .3 I.2 I.5
I.3 1.3
1.2 1.8
2.4 2.2 2.3
1.4 I.4 I.4
1.6 1.5 I .6
5.6 0.24
2.2
3.8
-
-
The diameter d was calculated from the pore conductance in
I M KCI according to A = g a r ’ / / (using u = 110 mS cm-l and I =
7.5 nm). The nomenclature for the E . coli porins was taken from Lugtenberg (1981).
larger than the diameters of the other porin pores. Assuming that the porin pores are filled with a solution of the same specific conductivity as the external solution and assuming a pore length of 7.5 nm [corresponding to the thickness of the outer membrane (Di Rienzo et al., 1978; Nikaido, 1979a)], according to the equation
A
= o.rrr2/1
(3)
the average pore diameter d ( = 2 r ) and the cross section can be calculated. Table I1 shows the diameter and cross section for porins from different gram-negative bacteria, calculated from the conductance of the pores in I M KCl (a= 110 mS cm-I). The values for the diameter are consistent with the results of the reconstituted vesicles, in which hydrophilic solutes of molecular weights up to 600 ( E . coli; Nakae, 1976), 700 (S. typhitnurium; Nakae, 1975), and 6000 (P. aeruginosa; Hancock ef al., 1979) were found to be permeable through the porin channels. The properties of the porin P channel from P . aeruginosa outer membrane are quite different from those of the other porins. Whereas most porins have only a limited selectivity, the porin P channel appears highly anion selective, with PJP, > 100. The decreasing single-channel conductance for larger halides indicates a small selectivity filter with a diameter
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
215
-0.6 nm, compared with a diameter of at least 1.2 nm for the other porins. Furthermore, the single-channel conductance of the protein P channel saturates for salt concentrations greater than 0.3 M . Taking all these findings together, it is obvious that further studies of the porin P channel as a model for anion transport through channels will be of special interest. The outer membrane of gram-negative bacteria acts as a molecular filter for hydrophilic solutes (Di Rienzo et a f . , 1978; Nikaido, 1979a). The results obtained for lipid bilayer membranes support this view. In particular, we found no gating of the porin pores and found that the pores had a long lifetime (of the order of minutes). Only the porin F pores from P . aeruginosa showed a much shorter lifetime (50-100 msec) if total outer membrane, instead of purified porin, was present in the experiments. This is presumably related to the high antibiotic resistance of this organism as has been discussed elsewhere (Angus et a/., 1981). In this case also, a voltage-controlled gating process could not be detected. Furthermore, the vesicle permeability assay (Mancock and Nikaido, 1978; Hancock et a / . , 1979; Nakae, 1975, 1976) is consistent with the presence of open pores in the absence of applied voltage, a fact which supports the results obtained from lipid bilayer membranes. These findings are in contrast to the results reported by Schindler and Rosenbusch (1978, 1981), who studied porin pores from E . c d i with “folded” lipid bilayer membranes formed from reconstituted vesicles. In these experiments no pores were found to be open after membrane formation at zero voltage, and large membrane potentials were needed to switch on ( V , = 100 mV) and switch off ( V , L IS0 mV) the porin pores. However, the control of membrane permeability by voltage-gated pores in the outer membrane of gram-negative bacteria, as suggested by Schindler and Rosenbusch (1978), is difficult to understand. The channel density in the outer membrane of grarn-negative bacteria is about lo’* pores/cm2 (Di Rienzo et af., 1978; Nikaido, 1979b) and the time constant of the membrane is very small under normal conditions (10 nsec at 0. I M salt; Benz c’t a / . , 1980). Any membrane patential will drop immediately to zero, and the Donnan potentials, which have been reported to be about 20-30 mV across the outer membrane of S . typhimirrium and E. coli (Stock et al., 1977), are far too small to reach 100 mV. We believe, therefore, that something other than voltage-gated pores must be responsible for the voltage effects observed by Schindler and Rosenbusch (1978). One explanation could be that the porin molecules are located only in one monolayer and do not penetrate the “folded” membranes. The high electric field could facilitate the insertion of the proteins into both monolayers of the folded membrane and this process could open the pores. Another
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explanation could be that for unknown reasons the folded membranes do not contain any active porin after their formation. The attached vesicles, which contain porin, could fuse with the membranes under the influence of the high electric field. The latter possibility would also explain the large conductance jumps observed by Schindler and Rosenbusch (1978, 19811. Similar jumps have been reported for the fusion of porin-containing vesicles with planar lipid bilayer membranes (Cohen et al., 1982). It is still an open question as to whether one porin trimer contains three pores (Nikaido, 1979b: Schindler and Rosenbusch, 1978)or only one pore (Benz et al., 1978a; Di Rienzo et al., 1978). The conductance data presented in Table I suggest that one trimer contains only one pore. For the case of three pores in a trimer, the pore diameter as calculated from the conductance data would be simply too small to account for the permeation of large hydrophilic solutes through the porin pores. The case for one pore per trimer is also supported by the finding that porin monomers have been found to be inactive in reconstitution experiments with vesicles (Nakae et al., 1979). The amino acid composition and the sequence of all three porins of E . coli outer membrane (OmpF, OmpC, and PhoE) have been determined in a number of recent publications (Chen et al., 1979; Inokuchi et al., 1982; Mizuno et al., 1983; Overbeeke et al., 1983). The sequences of all three porin monomers are not particularly hydrophobic. Only a limited number of subsequences with more than three consecutive hydrophobic amino acids have been found in this protein. Furthermore, it is evident from the sequences that the structural genes for the three porins evolved from a common ancestral gene (Mizuno et al., 1983). This shows that the arrangement of the polypeptide chain in the tertiary and quaternary structure is responsible for the pore formation (Chen et al., 1979; Palva and Randall, 1978). The fact that E. coli porin has been shown to contain a large amount of p-sheet structure supports this hypothesis (Nakae et al., 1979). The cation selectivity of the OmpF and the OmpC porin pores arises presumably from the excess of negatively charged amino acids (Inokuchi et al., 1982; Mizuno et al., 1983), whereas the anion selectivity of the PhoE pore may be caused by the appearance of additional lysines in the sequence (Benz et al., 1984a; Overbeeke et al., 1983). The study of porin channels from gram-negative bacteria offers an elegant way to investigate the structure-function relationship of pores. Porin trimers are usually very stable and allow chemical modifications without damage to protein structure (Tokunaga et al., 1981). In particular, it is possible to change the ionic selectivity of porin channels by such chemical modification (Benz et al., 1984b). Of further interest is the study of anion transport through the selective porin from P . aeruginosm (Benz et d.,
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
217
1983; Hancock c’t d.,1982) and the influence of chemical modification on anion transport (Hancock ct a / . , 1983). REFERENCES Angus, B. L., Carey, A. M.. Caron, D. A. Kropinski, A. M. B., and Hancock, R. E. W. (I98 1). Outer membrane permeability in Pseudomonus ueruginosu: Comparison of a wild-type with an antibiotic-supersusceptible mutant. Antinzicrob. Agents Chemother. 21, 299-309. Benz, R., and Hancock, R. E. W. (1981). Properties of the large ion permeable pores formed from protein I of P.sr14domoncr.s crrrrrginosu in lipid bilayer membranes. Bioc.hinl. Biophys. Actu 646, 298-308. Benz, R., Janko, K., Boos, W., a i d Lauger P. (1978a). Formation of large ion-permeable membrane channels by the matrix protein (porin) of Escherichiu coli. Biochim. Biophys. Actu 511, 305-319. Benz, R., Boehler-Kohler. B. A . , Dieterle, R., and Boos, W. (1978b). Porin activity in the osmotic shock fluid of E.sc~heric~hiu i,oli. J . Bwteriol. 135, 1080- 1090. Benz, R.,Janko, K., and Lauger, P. (1979). Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli. Biochim. Biophys. Actu 551, 238-247. Benz, R., Ishii, J., and Nakae, T. (1980). Determination of ion permeability through the channels made of porins from the outer membrane of Salmonella typhimurium in lipid bilayer membranes. J . Membr. B i d . 56, 19-29. Benz, R., Gimple, M., Poole, K., and Hancock, R. E. W. (1983). An anion selective channel from the P.seitdomonus urrrrginosu outer membrane. Biochirn. Biopl7ys. A i m 7-W, 387-390. Benz. R.. Darveau. R. P.. and Hancock, R. E. W. (1984~1).Outer membrane protein PhoE from Escherichiu coli forms anion-selective pores in lipid bilayer membranes. Eur. J . Biochem. (in press). Benz, R., Tokunaga, H., and Nakpe, T. (1984b). Properties of chemically modified porin from Eschrrichiu coli in lipid bilayer membranes. Biochim. Biophys. Actu 769, 348356.
Chen, R., Kramer, C.. Schmidmayer, W., and Henning, U . (1979). Primary structure of major outer membrane protein 1 of Escherichiu coli B. Proc. Nut/. Arad. Sci. U . S . A . 76, 5014-5017. Cohen, F. S . , Akabas, M. H., and Finkelstein, A. (1982). Osmotic swelling of phospholipid vesicles causes them to fuse with a planar phospholipid bilayer membrane. Science 217, 458-460. Di Rienzo, J. M., Nakamura, K., and lnouye, M. (1978). The outer membrane of gramnegative bacteria: Biosynthesis. assembly and function. Annu. Reu. Biochem. 47,481532. Hancock, R. E. W., and Nikaido. H. (1978). Outer membranes of gram-negative bacteria XIX. Isolation from Pseudomonas ueruginosa PA 01 and use in reconstitution and definition of permeability barrier. J. Bacteriol. 136, 381-390. Hancock, R. E. W., Decad, G . M., and Nikaido, H. (1979). Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonns ueruginosa PA 01. Biochim. Biophys. Acta 554, 323-331. Hancock, R. E. W., Poole, R. K , , and Benz, R. (1982). Outer membrane protein P of Pseudomonas ueruginosu regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J . Bucteriol. 150, 730-738.
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Hancock, R. E. W., Poole, K., Gimple, M., and Benz, R. (1983). Modification of the conductance, selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation. Biochim. Biophys. Acfa 735, 137-144. Inokuchi, K., Mutoh, N., Matsuyama, S . , and Mizushima, S. (1982). Primary structure of the OmpF gene that codes for a major outer membrane protein of Escherichia coli K12. Nucleic Acids Res. 10, 6957-6968. Lauger, P. (1973). Ion transport through pores: A rate-theory analysis. Biochim. BiophyJ. ACIU 311, 423-44 I . Lugtenberg, B. (1981). Composition and function of the outer membrane of Escherichia coli. Trends Biochem. Sci. 6, 262-266. Miller, C., and Racker, E. (1976). Cat+-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J . Membr. Biol. 30, 283-300. Miller, C., Arvan, P.. Telford, J . N., and Racker, E. (1976). Ca++-inducedfusion of proteoliposomes: Dependence on transmembrane osmotic gradient. J . Membr. Biol. 30,271 282. Mizuno, M., Chou, M.-Y., and Inouye, M. (1983). A comparative study on the genes for three porins of the Escherichia coli outer membrane: DNA sequence of the osmoregulated ompC gene. J . Biol. Chem. 11, 6932-6940. Montal, M., and Mueller, P. (1972). Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. U . S . A . 69, 3561-3566. Nakae, T. (1975). Outer membrane of Salmonella iyphimurium: Reconstitution of sucrosepermeable vesicles. Biochem. Biophys. Res. Commun. 64, 1224-1230. Nakae, T. (1976). Identification of the outer membrane protein of Escherichia coli that produces transmembrane channels in reconstituted vesicle membranes. Biochem. Biophys. Res. Commun. 71, 877-889. Nakae, T., and Ishii, J . (1978). Transmembrane permeability channels in vesicles reconstituted from single species of porin from Salmonella fyphimurium. J . Bacteriol. 133, I4 12-14 18. Nakae, T., Ishii, J . , and Tokunaga, M. (1979). Subunit structure of functional porin oligomers that form permeability channels in the outer membrane of Escherichia coli. J . Biol. Chem. 254, 1457-1461. Nikaido, H. (1979a). Nonspecific transport through the outer membrane. In “Bacterial Outer Membranes” (M. Inouye, ed.), pp. 361-407. Wiley (Interscience), New York. Nikaido, H. (197913). Permeability of the outer membrane of bacteria. Angew. Chem., f n i . Ed. Engl. 18, 337-349. Nikaido, H . , and Nakae, T. (1979). The outer membrane of gram-negative bacteria. Adu. Microb. Physiol. 20, 163-250. Overbeeke, N., Bergmans, H.. van Mansfield, F., and Lugtenberg, B. (1983). Complete nucleotide sequences of phoE, the structural gene for the phosphate lirnitation-inducible outer membrane pore protein of Escherichia coli K-12. J . Mol. Biol. 163, 513-532. Palva, E. T., and Randall, L. L. (1978). Arrangement of protein I in Escherichia coli outer membrane: Cross-linking studies. J . Bacferiol. 133, 279-286. Rosenbusch, J. P. (1974). Characterization of the major envelope protein from Escherichia coli. J . Biol. Chem. 249, 8019-8029. Schindler, H . , and Rosenbusch, J . P. (1978). Matrix protein of Escherichia coli outer membranes forms voltage-controlled channels in lipid bilayers. Proc. Narl. Acad. Sci. U . S . A . 75, 3751-3755.
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Schindler, H., and Rosenbusch, J. P. (1981). Matrix protein in planar membranes: Clusters of channels in a native environment and their functional reassembly. Prnc. Nut/. Acud. Sci. U.S.A. 78, 2302-2306. Stock, J . B., Rauch, B., and Roseman, S . (1977). Periplasmic space in Salmonelku ryphimurirtm and Escherichia coli. J . B i d . Chem. 252, 7850-7861. Tokunaga, H., Tokunaga, M . , and Nakae, T. (1981). Permeability properties of chemically modified porin trimers from Escherichia coli B. J . B i d . Chem. 256, 8024-8029. Tommassen, J., and Lugtenberg, B. (1980). Outer membrane protein e of Escherichiu coli K-12 is co-regulated with alkaline phosphatase. J . Bacterial. 143, 151-157.
Structure and Selectivity of Porin Channels R . BENZ Dcpartmcnt of Biology Univcrsity of Konstanz Konstanz, Fcdcral Republic of Gcrmuny
I . Introduction. ...... 11. Reconstitutio rins in .............. A. Addition of Porin to the Aqueous Phase.. . . . . B. Membrane Formation from C. Fusion of Reconstituted Ve 111. Single-Channel Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Ionic Selectivity of Porin Channels . . . . . . lective Channel V. Properties of the .............. Outer Membrane VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
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200 200 203 204 205 207 209 213 211
INTRODUCTION
The cell wall of gram-negative bacteria consists of three different layers, the inner membrane, the peptidoglycan layer, and the outer membrane (Di Rienzo et al., 1978; Nikaido, 1979a).The inner membrane acts as a real permeability barrier and contains, in addition to the respiration chain, a large number of different transport systems for substrates, whereas the peptidoglycan layer prevents lysis of the bacterium. The outer membrane seems to act a5 a molecular filter for hydrophilic wbstances and as a barrier for hydrophobic molecules (Nikaido, 1979b; Nikaido and Nakae, 1979). The sieving property of the outer membrane of gram-negative bacteria resides in a defined exclusion limit for hydrophilic solutes. The permeability of the outer membrane I S associated with the presence in the outer bacterial membrane of a class of major proteins called matrix proteins (Rosenbusch, 1974) or porins (Nakae, 1975, 1976). 199 Copyright CJ 1984 by Academic P r e s . Inc All right\ ot reproduction in dny form reserved
ISBN n-12-153121-2
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Porins have been isolated from the outer membrane of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa (Hancock et al. , 1979, 1982; Nakae, 1976; Nakae and Ishii, 1978). They form trimers in dodecyl sulfate that are usually stable. Experiments on reconstituted vesicles in the presence of such porin trimers from enteric bacteria have shown that the maximum molecular weight for permeable substances in the case of E. coli and S. typhimurium is between 600 and 700, whereas molecules with molecular weights up to 6000 can penetrate vesicles reconstituted in the presence of porin F from P. aeruginosa (Hancock et al., 1979; Nakae, 1976; Nakae and Ishii, 1978). Experiments on reconstituted vesicles provide valuable information on the presence and the size of the pores formed by a porin. More detailed information about the pore interior and pore selectivity can be obtained from experiments on lipid bilayer membranes. The reconstitution of the porins into lipid bilayers and the properties of the porin channels from E . coli, S. typhimurium, and P. aeruginosa in lipid bilayer membrane are described in this article. In lipid bilayer membranes the porins of these gram-negative bacteria form, in general, large water-filled pores with a poor selectivity for ions. However, there are exceptions. Protein P from P . aeruginosa outer membrane forms highly anion-selective channels in lipid bilayer membranes (Benz et al., 1983; Hancock et al., 1982). II. RECONSTITUTION OF PORINS INTO LIPID BILAYER MEMBRANES A. Addition of Porin to the Aqueous Phase
Three different methods have been used successfully to reconstitute porins into lipid bilayer membranes. Firsi, detergent-solubilized porin is directly added to the aqueous phase bathing a membrane (Benz et al., 1978a); second, the lipid bilayer membrane is formed from reconstituted vesicles according to the Montal-Mueller method (Schindler and Rosenbusch, 1978); and third, the porin is inserted into the planar bilayer via fusion of the reconstituted vesicles with the membrane (Cohen et al., 1982). The simplest method consists of the addition of purified porin from a stock solution containing. 0.1% dodecyl sulfate or Triton X-100 to the aqueous phase bathing a black lipid bilayer membrane (Benz et af., 1978a). Figure I shows such an experiment. Porin from S. typhimuriurn strain SH 5551 (M, 40,000) was added in a final concentration of 100 ng/ml
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
10-8
201
3 0 10 20 30 LO t (rnin)
FIG.I . Specific membrane conductance as a function of time after addition of 100 nglml trimers of S. tvphinirrrirrm strain SH 5551 ( M ,40.000) to a black membrane from egg phosphatidylcholineln-decane (closed cir'cles, arrow). The open circles represent a control experiment in which only 10 pg/ml SUS was added to another membrane. I M KCI. 25°C.
to a black membrane from egg phosphatidylcholine/n-decane.After an initial lag of 4 minutes, presumably due to diffusion of the protein through unstirred layers, the conductance increased by about three orders of magnitude within about 30 minutes. Only a slight additional increase (as compared with the initial one) occurred after that time. It has to be stressed, however, that in the presence of all porins the membrane conductance increases continuously until membrane breakage. It is interesting to note that detergents had only a small influence, if any, on porin reconstitution. This was shown by using various detergents and detergent-free porin from osmotic shock solution (Benz et id., 1978a,b). Porin trimers dissociated into monomers were found to be inactive in lipid bilayer membranes and reconstituted vesicles (Nakae et a / ., 1979). Since in all the experiments with porins a steady conductance level could not be reached, the dependence of the conductance on various parameters was difficult to determine. A meaningful comparison was possible, however, on the basis pf experiments similar to those presented in Fig. 1, using the conductance value at a fixed time after addition of the protein. Figure 2 shows the influence of the membrane composition on the incorporation of the porins from S . typlzirnrrrium strain TA 1014 ( M , 38,000, 39,000, and 40,000) into the membranes. For membranes made from oxidized cholesterol or monoolein, the conductance is about two to
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Protein concentration (ng/rnl)
FIG.2. Specific membrane conductance A as a function of the concentration of trimers from the Salmonella strain TA 1014 ( M , 38,000, 39,000, and 40,000) in the aqueous phase; 1 M KCI; T = 25°C. Less than 5 pg/ml SDS was present in the aqueous solutions. The membranes were formed from different lipids dissolved in n-decane. The measurements were performed 20 minutes after blackening of the membranes. (0) Oxidized cholesterol; ( W monoolein; ( 0 )brain phosphatidylserine; (0) egg phosphatidylcholine; ( X ) egg phosphatidylethanolamine.
three orders of magnitude larger than for membranes made from phospholipids. A similar “lipid specificity” was also found for the porins from E . coli and P. aeruginosa (Benz et al., 1978a; Benz and Hancock, 1981). It is obvious that many conductive units were incorporated into lipid bilayer membranes in the experiments described above. This allows investigation of the current-voltage behavior of membranes containing a large number of porin trirners. Examples of current-voltage curves of two membranes made from oxidized cholesterolln-decane in the presence of porin trimers of 3. typhimurium strain HN 6017 ( M , 38,000) are given in Fig. 3. The observed current was a linear function of applied voltage up to at least 150 mV. Even for an application of 100 mV for more than 30 seconds no current decrease occurred, but sometimes a slight increase was found-presumably caused by the incorporation of the porin into the membranes, facilitated by the high electrical field. Linear current-voltage relationships were also found for porins from E . coli and P. aeruginosa (Benz et al., 1978a; Benz and Hancock, 1981; Hancock et al., 1982). The results strongly suggest that no voltage is required to initiate the single conductance unit, i.e., the pores are not voltage gated if the porin molecules are reconstituted by addition to the aqueous phase.
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
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FIG.3. Current vs voltage characteristics of two membranes from oxidized cholesterol/ n-decane doped with I ng/ml porin ttimers from S. fypypAimitrium strain H N 6017 ( M , 38,000). The aqueous phase contained 1 M KCI and 50 ng/ml sodium dodecyl sulfate; 7 = 25°C.
B. Membrane Formation from Reconstituted Vesicles
Schindler and Rosenbusch (1978) used an alternative method for incorporation of porin molecules into lipid bilayer membranes. Vesicles reconstituted from lipids and porin are added to the aqueous phases on both sides of a thin Teflon foil having a small circular hole (100-200 pm in diameter) above the initial water level. Monolayers form on the surfaces of the two aqueous compartments. It is suggested by Schindler and Rosenbusch (1978) that these monolayers have the same composition as the vesicles. The water levels on both sides of the foil are now raised above the hole and a “folded” membrane is formed across the hole (Montal and Mueller, 1972). No pores are observed in the membrane immediately after formation at low membrane voltages. Membrane potentials around 100 mV are needed to induce the pores in steps larger than one single conductive unit (Schindler and Rosenbusch, 1978, 1981). Figure 4 shows such an experiment taken from Schindler and Rosenbusch (1978). The initial jumps indicate the formation of pore multimers in 0.1 M NaCl at 240 mV, whereas the subsequent decay is due to inactivation of the single conductive unit, caused by the high voltage. Schindler and Rosenbusch (1978) suggested the existence of three pores in a porin trimer. According to their view, all three pores should activate together but inactivate separately (see also Fig. 4). As a consequence of their observations, Schindler
i
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0
m
05 0
4 0‘
2
10
15
1 20
Time [minl
FIG.4. Initiation of conductance in a porin-containing membrane made from monolayers. The left side shows large current .jumps which appear at 240 m V membrane potential in 0. I M NaCI. Each large increment was followed by a decay in small steps (0.14 pS) as shown on the right side of the figure. (Taken with permission from Schindler and Rosenbusch, 1978.)
and Rosenbusch (1978) proposed a voltage control of the porin pores in the outer membranes of gram-negative bacteria. C. Fusion of Reconstituted Vesicles with Planar Lipid Bilayers
The third possibility of incorporation of porin molecules into planar lipid bilayer membranes is through fusion of reconstituted vesicles with planar bilayers. Miller and Racker (1976) and Miller et a1 (1976; and see Miller et ul., this volume) showed that cytochrome oxidase vesicles and vesicles from sarcoplasmic reticulum could be fused with planar bilayers in the presence of calcium ions. The calcium ions are most probably needed for the close apposition of the vesicles to the membranes. The yield of fusion could be drastically increased if an osmotic gradient hyperosmotic on the cis side (the side of the addition of the vesicles) were applied across the planar bilayer. The osmotic gradient results in a water flux toward the cis side. This water flux for unknown reasons accelerates the fusion process of the lipid vesicles with the planar bilayer. Recently, Cohen et al. (1982) used this method to incorporate porin molecules into planar bilayers. Upon addition of calcium ions to the cis side and establishing an osmotic gradient hyperosmotic at the cis side, the fusion process begins as indicated by a stepwise increase of the membrane current. The fusion process stops after the osmotic gradient is removed by addition of the osmotic active substance to the trans side. Furthermore, Cohen et ul. (1982) could show that water flux into the reconsti-
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OF PORIN CHANNELS
tuted vesicles without any gradient across the planar bilayer also increases the yield of fusion. It is interesting to note that the fusion process resulted immediately in open pores in the lipid bilayer membranes and that no voltage-induced activation of pores was observed by Cohen et ul. (1982). Only a minor inactivation was found at high voltages prior to membrane breakage.
I II. SI NOLE-CHAN NEL AN ALYSl S The addition of small amounts (1-10 ng/ml) of porins to the aqueous phase bathing a membrane of small surface resulted in a stepwise increase of the membrane current at H given voltage. These current jumps were not observed when only the detergents dodecyl sulfate or Triton X-100 were added to the aqueous phase in the same concentration as the porin. Figure 5 shows an experiment using porin from E . coli. As can be seen from Fig. 5 all steps were directed upward, whereas downward steps are only rarely observed. Similar results are also found for the porins from S. typhirnuriurn and P . aeruginosu. The lifetime of all porin pores was at least I minute, as could be seen from records extending over long times. Only porin F pores observed in the presence of total outer membrane from P . ueruginosa (Hancock and Nikaido, 1978) had a much shorter lifetime, on the order of 50-100 msec, although the absolute level of the pore conduc-
0.5nS 25 pA
FIG.5. Stepwise increase of the membrane current in the presence of 0. I ng/ml porin from E. coli K12 added to the q u e o u s phase containing 0.1 M NaCl; T = 25°C. The membrane was formed from egg phosphatidylcholineln-decane;V , = SO mV. The record starts at the left end of the lower trace and continue5 in the upper trace.
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3 :0 ;1 P
0
0
li
0.1
1
L 02
03
OL
A (ns)
FIG.6. Histogram of the conductance fluctuations observed with membranes from egg phosphatidylcholineln-decanein the presence of porin from E . coli K12, 0.1 M NaCI; T = 25°C. n, the number of single steps; V , = 50 mV.
tance was not changed as compared with purified porin F (Benz and Hancock, 1981). Most of the conductance fluctuations obtained with various porins were not uniform in size but were distributed over similar ranges. A histogram of the conductance steps measured with porin from E . coli is shown in Fig. 6. The fluctuations for the E . coli porin varied about fourfold. Similar distributions were found for all porins. Only the recently discovered anion channel formed by porin P from P . aeruginosa gave a narrow distribution of the conductance fluctuations (Benz et al., 1983; Hancock et al., 1982). Single-channel measurements in the presence of the porin were performed with a variety of different salts and concentrations. From records similar to those given in Fig. 5 the average conductance increment A was obtained by measuring a sufficient number (at least 50) of individual events. For all porins described in this review (except porin P from P . aeruginosa; Benz et al., 1983) the average pore conductance A was a linear function of the specific conductance u of the aqueous phase, i.e., the ratio Alu varied only little in contrast to A, which varied by two orders of magnitude. This is also reflected in Fig. 7, where the average pore conductance A for the porin F of P . aeruginosa and a porin of E . coli are given as a function of u.The data points could be fitted with straight lines. The same is valid for the porins from S . typhiniurium, although the
207
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
001.
0 FIG. 7. Average pore conductance A of porin from E . coli K I? (open circles) and protein F from P . a ~ r u g i n o s a(closed circles) given as a function of the specific conductance of the corresponding aqueous salt solution; T = 25°C.
ratio Alu showed a much larger variation for E. c d i and S . typhimrrrirrm than for P . creri4g:ino.s~porin F. This can be explained by the larger diameter of the P . uerrrginosu pore. which is also consistent with the larger value of Alu (Fig. 7) and with a vesicle permeability assay using carbohydrates of different sizes (Hancock and Nikaido, 1978; Nakae, 1976). I t is interesting to note that even large organic cations and anions such as Tris', N(CH2CH3);, and HEPES- were able to pass through the porin pores with little or no interaction with the pore interior.
IV. IONIC SELECTIVITY OF PORlN CHANNELS Further information on the structure of the conductance pathway created by the different porins may be obtained by studying the ionic selectivity of the pores using zero-current potential measurements. Figure 8 shows the results of such experiments obtained for E. coli porin oligomers (which contained two different porins called OmpF and OmpC) in mcmbranes from oxidized cholesterolln-decane. The zero-current potential was found to be positive on the more dilute side of the membrane. This indicated that porin oligomers from E. coli form cation-selective pores in lipid bilayer membranes. From the measured V , and the concentration gradient c"Ic' across the membrane, the ratio P J P , of the permeabilities
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c”/c‘
FIG.8. Zero-current membrane potential V , as a function of the ratio of the salt concentrations on both sides of membranes containing porin from E . coli K12. The membranes were made from oxidized cholesterolln-decane; T = 25°C. V , was positive on the more dilute side of the membrane, c’ = lo-* M .The lines were drawn according to the GoldmanHodgkin-Katz equation with the specified values of the permeability ratio P,IP,. ( x ) KCI; (@) NaCI.
P , for cations and Pa for anions was calculated according to the GoldmanHodgkin-Katz equation: RT PCdr+ Pact V , = - In F Pccr t Pact’ where R , T , and F have their usual meanings. Table I shows the permeability ratios P J P , for the different porins with KCI as a salt in the aqueous phase. The observed slight selectivity for cations or anions may be explained by the presence of negative or positive charges, respectively, in or near the pores. The Pho E porin pore is induced in E . coli outer membrane under conditions of phosphate deprivation (Tommassen and Lugtenberg, 1980). The assumption that charged groups are responsible for the selectivity is supported by the pH dependence of the selectivity. The permeability ratio P J P , for the porin pore decreases in the presence of a NaCl gradient from 2.8 at pH 9 to 1.2 at pH 3 (Benz et al., 1979). Further support arises from selectivity changes of chemically modified porin. For example, porin OmpF from E . coli completely loses its cation
209
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
'TABLE 1
ZERO-CURRENT POTFNTIAI s V,,, FOR
THE
PRESENCE O F A IO-POI
D
DIFFERENT PORINSI N KCI GRADENr"
Porin
pH
V,,,( m V )
E . co/i OmpF/OmpC (la/lb) OmpF/OmpC (lailb) OmpF/OmpC (Ia/lb) PhoE (Ic)
h 3 9 h
27 t 3 -5 t 2 30 3 -25t5
S. rypliimitrirtm M, 40,000
h
24 t 2
3.2 t 0,s
20
2.4 2 0.4 10
P.
"
3.8 t 0.9 0.80 0. I 4.6 -t 0.9 0.29 t 0.08
*
trerllgitloscl
F P
6 h
'' 4
-57 + 4
V , is the electrical potential of the dilute side (lo-? M ) minus the potentilt1 of the concentrated side (10.' M ) . The membranes were formed from egg phosphatidylcholineln-decane or diphytanoylphosphatidylcholine/n-decane;T = 25°C. The ratios of the permeability P, (cation) and P, (anion) were calculated from the Goldman-Hodgkin-Katz equation. The nomenclature for the E . c d i porins was taken from Lugtenberg (1981).
selectivity after amidation, i.e., PJP,, = 1 (Tokunaga e t a / . , 1981; Benz c't a / . , 1984b). V.
PROPERTIES OF THE ANION-SELECTIVE CHANNEL FROM Pseudornonas oeruginosa OUTER MEMBRANE
The outer membrane of P. u~rrcginosrrobviously has very special sieving properties, which make this organism quite resistant to most antibiotics (Angus rt u/., 1981; Hancock rt d . , 1982). One of these sieving properties is the relatively short lifetime of the porin F channel. Presumably also present in the outer membrane is protein P ( M , 48,000), which is induced when P. creruginow is grown on media with phosphate limitations. Protein P forms anion-selective channels in lipid bilayer membranes. Figure 9 shows zero-current membrane potential measurements on dioleylphosphatidylchol/nemembranes in the presence of protein P and a KCI gradient. The zero-current membrane potential was negative on the more dilute side and could be fitted reasonably well to the Goldman-Hodgkin-Katz equation assuming either a permeability ratio PJP, = 0 (Nernst equation, full line) or PJP,, = lo-* (broken line). According to
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C'/d'
FIG. 9. Zero-current membrane potentials V , of diphytanoylphosphatidylcholine/n-decane membranes measured as function of a KCI gradient in the presence of protein P from P . ueruginosu; T = 25"C, V,,,was negative on the more dilute side of the membrane, c'' = lo-* M . The lines were drawn according to the Goldman-Hodgkin-Katz equation assuming P J P , = 10-' (broken line) or PJP., = 0 (solid line); T = 25"C, pH 6.
this result, chloride permeates through the porin P channel at least 100 times faster than does the potassium ion (Benz et ul., 1983). The current fluctuations of the porin P channel from P . ueruginosu outer membrane are found to be fairly homogeneous, in contrast to the steps in current observed with porins from Salmonella (Benz et al., 1980) or E . coli (Benz et al., 1978a). Figure 10 shows current fluctuations observed with protein P on a diphytanoylphosphatidylcholine membrane. There all current steps were directed upward, closing pores being only rarely observed. This indicates a long lifetime, as is found for the other porin pores. The lifetime of single steps was found to be independent of voltage, and linear current-voltage relationships were observed up to 200 m V for the protein P channels. The single-channel conductance of the porin P channel was found to be independent of the type of cation present in the aqueous phase. For a 100 mM chloride solution the single-channel conductance was 160 pS, irrespective of the size and nature of the cation. A much larger variation of the single-channel conductance A was obtained if the anions were varied. Figure 1I shows the dependence of the single-channel conductance A of
-
21 1
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
-
1
100 pA
2nS
FIG. 10. Single-channel record of a diphytanoylphosphatidylcholine/n-decane membrane in the presence of 10 ng/ml protein P from P . aerrcginnsci and 1 M KF in the aqueous phase. A voltage of 50 mV was applied through calomel electrodes with salt bridges; T = 25°C.
100 mM salt solutions on the size of the halides. The corresponding cation was in all cases potassium. The conductance appears to be highest for chloride and lowest for iodide. Besides the halides, a variety of other anions, such as nitrate and nitrite, also permeate through the porin P channel, whereas in the presence of salts with large organic anions, such as HEPES- (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid), no conductance fluctuations could be observed at all. The origin of the anion selectivity of the porin P pore from P . ueruginostr outer membrane is, therefore, presumably a selectivity filter with a diameter of -0.6 nm containing at least one positive charge. This structure accounts also for the concentration dependence of the single-channel conductance. Figure 12 shows the dependence of the single-channel conductance on the KCl concentration c in the aqueous phase. A linear conductance-concentration relationship was observed only with KCl concentrations as small as 1 and 10 mM. The single-channel conductance approached saturation for higher salt concentrations, and no further conductance increase occurred above 0.3 M KCI. The conductance concen-
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21 2
3m/
Radius (nml
FIG.11. Single-channel conductance of the porin P channel in diphytanoylphosphatidylcholineln-decane membranes as a function of the ion radius of the anion according to Pauling. 50 mV was applied to the membranes through calomel electrodes with salt bridges; T = 25°C.
Id'
1
FIG. 12. Single-channel conductance of the porin P channel from P . aeruginosa outer membrane as a function of the potassium chloride concentration in the aqueous phase. The membranes were formed from diphytanoylphosphatidylcholineln-decane. 50 mV was applied to the membranes; T = 25°C. The solid line was drawn according to Eq. (2) with the values of Amax= 280 pS, and K = 20 M-I.
STRUCTURE AND SELECTIVITY OF PORIN CHANNELS
213
tration curve A,((.) given in Fig. 12 could be fitted easily assuming single occupancy of the pore (i.e,, one binding site for anions in the pore), using the following equation [Lauger, 1973; see also Eq. (17) of Lauger, this volume]: &(c) = [cK/(I + ~ K ) ] h o , , , , , (2) with the binding constant K = 20 M-' and a maximum conductance of 280 pS (solid curve in Fig. 12). VI.
CONCLUSIONS
Porins isolated from the outer membrane of gram-negative bacteria are able to form ion-permeable pores in lipid bilayer membranes. Three different methods can be used for successful reconstitution of the porins into lipid bilayer membranes; the implications of the different methods are discussed in more detail below. Most investigations were performed by addition of the proteins to the aqueous phase. The insertion into the membranes is in this case presumably controlled by hydrophobic forces because the reconstitution rate shows no dependence on the ionic strength of the aqueous phase (Benz et al., 1978a, 1980). The reconstitution rate has been found to be strongly dependent on the type of lipid used for membrane formation. The conductance and the ionic selectivity of the single conductance unit, however, are independent of the nature of the lipid. We believe, therefore, that the reconstitution rate does not reflect a specific lipid-protein interaction. Moreover, it seems that the insertion of the trimers into the membranes is governed by a kinetic process, for example the replacement of 100 lipid molecules (each of area 0.5 nm?) by one porin trimer (area 50 nm2; Nikaido, 1979a), which may need considerable energy (Benz ct al., 1980). The data presented here support the assumption that large water-filled pores are formed by most, but not all, porins. Let us first consider the large pores. These pores are permeable to the large organic ions Tris+, N(C2Hs):, and HEPES- without detectable interaction with the pore interior. Furthermore, pore conductance shows no saturation with increasing salt concentration in the aqueous phase, and the single-channel conductance was a linear function of the specific conductance of all salt solutions. The current-voltage characteristic was ohmic-which, too, is expected for a wide unselective channel. Nevertheless there exist some differences in the single-channel conductances. The single-channel conductance of porin F from P . nevuginosa was considerably higher than conductances observed for porins from E. (di and S. fyphirnrrrirrm (Table 11). This indicates that the diameter of the porin F pore is considerably
214
R. BEN2 TABLE I1 COMPARISON OF THE PORESFORMED B Y PORINS OF GRAM-NEGATIVE BACTERIA“ Pore
E . coli OmpF (Ia) OmpC (Ib) PhoE (Ic) LamB S . typhimurium M , 38,000 M , 39,000 M , 40,000 P . aeruginosa F P (1
A (nS)
d (nm)
Cross section (nm’)
1.9 I .9 1.7 2.7
1.3 I .3 I.2 I.5
I.3 1.3
1.2 1.8
2.4 2.2 2.3
1.4 I.4 I.4
1.6 1.5 I .6
5.6 0.24
2.2
3.8
-
-
The diameter d was calculated from the pore conductance in
I M KCI according to A = g a r ’ / / (using u = 110 mS cm-l and I =
7.5 nm). The nomenclature for the E . coli porins was taken from Lugtenberg (1981).
larger than the diameters of the other porin pores. Assuming that the porin pores are filled with a solution of the same specific conductivity as the external solution and assuming a pore length of 7.5 nm [corresponding to the thickness of the outer membrane (Di Rienzo et al., 1978; Nikaido, 1979a)], according to the equation
A
= o.rrr2/1
(3)
the average pore diameter d ( = 2 r ) and the cross section can be calculated. Table I1 shows the diameter and cross section for porins from different gram-negative bacteria, calculated from the conductance of the pores in I M KCl (a= 110 mS cm-I). The values for the diameter are consistent with the results of the reconstituted vesicles, in which hydrophilic solutes of molecular weights up to 600 ( E . coli; Nakae, 1976), 700 (S. typhitnurium; Nakae, 1975), and 6000 (P. aeruginosa; Hancock ef al., 1979) were found to be permeable through the porin channels. The properties of the porin P channel from P . aeruginosa outer membrane are quite different from those of the other porins. Whereas most porins have only a limited selectivity, the porin P channel appears highly anion selective, with PJP, > 100. The decreasing single-channel conductance for larger halides indicates a small selectivity filter with a diameter
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
215
-0.6 nm, compared with a diameter of at least 1.2 nm for the other porins. Furthermore, the single-channel conductance of the protein P channel saturates for salt concentrations greater than 0.3 M . Taking all these findings together, it is obvious that further studies of the porin P channel as a model for anion transport through channels will be of special interest. The outer membrane of gram-negative bacteria acts as a molecular filter for hydrophilic solutes (Di Rienzo et a f . , 1978; Nikaido, 1979a). The results obtained for lipid bilayer membranes support this view. In particular, we found no gating of the porin pores and found that the pores had a long lifetime (of the order of minutes). Only the porin F pores from P . aeruginosa showed a much shorter lifetime (50-100 msec) if total outer membrane, instead of purified porin, was present in the experiments. This is presumably related to the high antibiotic resistance of this organism as has been discussed elsewhere (Angus et a/., 1981). In this case also, a voltage-controlled gating process could not be detected. Furthermore, the vesicle permeability assay (Mancock and Nikaido, 1978; Hancock et a / . , 1979; Nakae, 1975, 1976) is consistent with the presence of open pores in the absence of applied voltage, a fact which supports the results obtained from lipid bilayer membranes. These findings are in contrast to the results reported by Schindler and Rosenbusch (1978, 1981), who studied porin pores from E . c d i with “folded” lipid bilayer membranes formed from reconstituted vesicles. In these experiments no pores were found to be open after membrane formation at zero voltage, and large membrane potentials were needed to switch on ( V , = 100 mV) and switch off ( V , L IS0 mV) the porin pores. However, the control of membrane permeability by voltage-gated pores in the outer membrane of gram-negative bacteria, as suggested by Schindler and Rosenbusch (1978), is difficult to understand. The channel density in the outer membrane of grarn-negative bacteria is about lo’* pores/cm2 (Di Rienzo et af., 1978; Nikaido, 1979b) and the time constant of the membrane is very small under normal conditions (10 nsec at 0. I M salt; Benz c’t a / . , 1980). Any membrane patential will drop immediately to zero, and the Donnan potentials, which have been reported to be about 20-30 mV across the outer membrane of S . typhimirrium and E. coli (Stock et al., 1977), are far too small to reach 100 mV. We believe, therefore, that something other than voltage-gated pores must be responsible for the voltage effects observed by Schindler and Rosenbusch (1978). One explanation could be that the porin molecules are located only in one monolayer and do not penetrate the “folded” membranes. The high electric field could facilitate the insertion of the proteins into both monolayers of the folded membrane and this process could open the pores. Another
216
R. BENZ
explanation could be that for unknown reasons the folded membranes do not contain any active porin after their formation. The attached vesicles, which contain porin, could fuse with the membranes under the influence of the high electric field. The latter possibility would also explain the large conductance jumps observed by Schindler and Rosenbusch (1978, 19811. Similar jumps have been reported for the fusion of porin-containing vesicles with planar lipid bilayer membranes (Cohen et al., 1982). It is still an open question as to whether one porin trimer contains three pores (Nikaido, 1979b: Schindler and Rosenbusch, 1978)or only one pore (Benz et al., 1978a; Di Rienzo et al., 1978). The conductance data presented in Table I suggest that one trimer contains only one pore. For the case of three pores in a trimer, the pore diameter as calculated from the conductance data would be simply too small to account for the permeation of large hydrophilic solutes through the porin pores. The case for one pore per trimer is also supported by the finding that porin monomers have been found to be inactive in reconstitution experiments with vesicles (Nakae et al., 1979). The amino acid composition and the sequence of all three porins of E . coli outer membrane (OmpF, OmpC, and PhoE) have been determined in a number of recent publications (Chen et al., 1979; Inokuchi et al., 1982; Mizuno et al., 1983; Overbeeke et al., 1983). The sequences of all three porin monomers are not particularly hydrophobic. Only a limited number of subsequences with more than three consecutive hydrophobic amino acids have been found in this protein. Furthermore, it is evident from the sequences that the structural genes for the three porins evolved from a common ancestral gene (Mizuno et al., 1983). This shows that the arrangement of the polypeptide chain in the tertiary and quaternary structure is responsible for the pore formation (Chen et al., 1979; Palva and Randall, 1978). The fact that E. coli porin has been shown to contain a large amount of p-sheet structure supports this hypothesis (Nakae et al., 1979). The cation selectivity of the OmpF and the OmpC porin pores arises presumably from the excess of negatively charged amino acids (Inokuchi et al., 1982; Mizuno et al., 1983), whereas the anion selectivity of the PhoE pore may be caused by the appearance of additional lysines in the sequence (Benz et al., 1984a; Overbeeke et al., 1983). The study of porin channels from gram-negative bacteria offers an elegant way to investigate the structure-function relationship of pores. Porin trimers are usually very stable and allow chemical modifications without damage to protein structure (Tokunaga et al., 1981). In particular, it is possible to change the ionic selectivity of porin channels by such chemical modification (Benz et al., 1984b). Of further interest is the study of anion transport through the selective porin from P . aeruginosm (Benz et d.,
STRUCTURE AND SELECTIVITY OF PORlN CHANNELS
217
1983; Hancock c’t d.,1982) and the influence of chemical modification on anion transport (Hancock ct a / . , 1983). REFERENCES Angus, B. L., Carey, A. M.. Caron, D. A. Kropinski, A. M. B., and Hancock, R. E. W. (I98 1). Outer membrane permeability in Pseudomonus ueruginosu: Comparison of a wild-type with an antibiotic-supersusceptible mutant. Antinzicrob. Agents Chemother. 21, 299-309. Benz, R., and Hancock, R. E. W. (1981). Properties of the large ion permeable pores formed from protein I of P.sr14domoncr.s crrrrrginosu in lipid bilayer membranes. Bioc.hinl. Biophys. Actu 646, 298-308. Benz, R., Janko, K., Boos, W., a i d Lauger P. (1978a). Formation of large ion-permeable membrane channels by the matrix protein (porin) of Escherichiu coli. Biochim. Biophys. Actu 511, 305-319. Benz, R., Boehler-Kohler. B. A . , Dieterle, R., and Boos, W. (1978b). Porin activity in the osmotic shock fluid of E.sc~heric~hiu i,oli. J . Bwteriol. 135, 1080- 1090. Benz, R.,Janko, K., and Lauger, P. (1979). Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli. Biochim. Biophys. Actu 551, 238-247. Benz, R., Ishii, J., and Nakae, T. (1980). Determination of ion permeability through the channels made of porins from the outer membrane of Salmonella typhimurium in lipid bilayer membranes. J . Membr. B i d . 56, 19-29. Benz, R., Gimple, M., Poole, K., and Hancock, R. E. W. (1983). An anion selective channel from the P.seitdomonus urrrrginosu outer membrane. Biochirn. Biopl7ys. A i m 7-W, 387-390. Benz. R.. Darveau. R. P.. and Hancock, R. E. W. (1984~1).Outer membrane protein PhoE from Escherichiu coli forms anion-selective pores in lipid bilayer membranes. Eur. J . Biochem. (in press). Benz, R., Tokunaga, H., and Nakpe, T. (1984b). Properties of chemically modified porin from Eschrrichiu coli in lipid bilayer membranes. Biochim. Biophys. Actu 769, 348356.
Chen, R., Kramer, C.. Schmidmayer, W., and Henning, U . (1979). Primary structure of major outer membrane protein 1 of Escherichiu coli B. Proc. Nut/. Arad. Sci. U . S . A . 76, 5014-5017. Cohen, F. S . , Akabas, M. H., and Finkelstein, A. (1982). Osmotic swelling of phospholipid vesicles causes them to fuse with a planar phospholipid bilayer membrane. Science 217, 458-460. Di Rienzo, J. M., Nakamura, K., and lnouye, M. (1978). The outer membrane of gramnegative bacteria: Biosynthesis. assembly and function. Annu. Reu. Biochem. 47,481532. Hancock, R. E. W., and Nikaido. H. (1978). Outer membranes of gram-negative bacteria XIX. Isolation from Pseudomonas ueruginosa PA 01 and use in reconstitution and definition of permeability barrier. J. Bacteriol. 136, 381-390. Hancock, R. E. W., Decad, G . M., and Nikaido, H. (1979). Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonns ueruginosa PA 01. Biochim. Biophys. Acta 554, 323-331. Hancock, R. E. W., Poole, R. K , , and Benz, R. (1982). Outer membrane protein P of Pseudomonas ueruginosu regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J . Bucteriol. 150, 730-738.
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Hancock, R. E. W., Poole, K., Gimple, M., and Benz, R. (1983). Modification of the conductance, selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation. Biochim. Biophys. Acfa 735, 137-144. Inokuchi, K., Mutoh, N., Matsuyama, S . , and Mizushima, S. (1982). Primary structure of the OmpF gene that codes for a major outer membrane protein of Escherichia coli K12. Nucleic Acids Res. 10, 6957-6968. Lauger, P. (1973). Ion transport through pores: A rate-theory analysis. Biochim. BiophyJ. ACIU 311, 423-44 I . Lugtenberg, B. (1981). Composition and function of the outer membrane of Escherichia coli. Trends Biochem. Sci. 6, 262-266. Miller, C., and Racker, E. (1976). Cat+-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J . Membr. Biol. 30, 283-300. Miller, C., Arvan, P.. Telford, J . N., and Racker, E. (1976). Ca++-inducedfusion of proteoliposomes: Dependence on transmembrane osmotic gradient. J . Membr. Biol. 30,271 282. Mizuno, M., Chou, M.-Y., and Inouye, M. (1983). A comparative study on the genes for three porins of the Escherichia coli outer membrane: DNA sequence of the osmoregulated ompC gene. J . Biol. Chem. 11, 6932-6940. Montal, M., and Mueller, P. (1972). Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. U . S . A . 69, 3561-3566. Nakae, T. (1975). Outer membrane of Salmonella iyphimurium: Reconstitution of sucrosepermeable vesicles. Biochem. Biophys. Res. Commun. 64, 1224-1230. Nakae, T. (1976). Identification of the outer membrane protein of Escherichia coli that produces transmembrane channels in reconstituted vesicle membranes. Biochem. Biophys. Res. Commun. 71, 877-889. Nakae, T., and Ishii, J . (1978). Transmembrane permeability channels in vesicles reconstituted from single species of porin from Salmonella fyphimurium. J . Bacteriol. 133, I4 12-14 18. Nakae, T., Ishii, J . , and Tokunaga, M. (1979). Subunit structure of functional porin oligomers that form permeability channels in the outer membrane of Escherichia coli. J . Biol. Chem. 254, 1457-1461. Nikaido, H. (1979a). Nonspecific transport through the outer membrane. In “Bacterial Outer Membranes” (M. Inouye, ed.), pp. 361-407. Wiley (Interscience), New York. Nikaido, H. (197913). Permeability of the outer membrane of bacteria. Angew. Chem., f n i . Ed. Engl. 18, 337-349. Nikaido, H . , and Nakae, T. (1979). The outer membrane of gram-negative bacteria. Adu. Microb. Physiol. 20, 163-250. Overbeeke, N., Bergmans, H.. van Mansfield, F., and Lugtenberg, B. (1983). Complete nucleotide sequences of phoE, the structural gene for the phosphate lirnitation-inducible outer membrane pore protein of Escherichia coli K-12. J . Mol. Biol. 163, 513-532. Palva, E. T., and Randall, L. L. (1978). Arrangement of protein I in Escherichia coli outer membrane: Cross-linking studies. J . Bacferiol. 133, 279-286. Rosenbusch, J. P. (1974). Characterization of the major envelope protein from Escherichia coli. J . Biol. Chem. 249, 8019-8029. Schindler, H . , and Rosenbusch, J . P. (1978). Matrix protein of Escherichia coli outer membranes forms voltage-controlled channels in lipid bilayers. Proc. Narl. Acad. Sci. U . S . A . 75, 3751-3755.
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Schindler, H., and Rosenbusch, J. P. (1981). Matrix protein in planar membranes: Clusters of channels in a native environment and their functional reassembly. Prnc. Nut/. Acud. Sci. U.S.A. 78, 2302-2306. Stock, J . B., Rauch, B., and Roseman, S . (1977). Periplasmic space in Salmonelku ryphimurirtm and Escherichia coli. J . B i d . Chem. 252, 7850-7861. Tokunaga, H., Tokunaga, M . , and Nakae, T. (1981). Permeability properties of chemically modified porin trimers from Escherichia coli B. J . B i d . Chem. 256, 8024-8029. Tommassen, J., and Lugtenberg, B. (1980). Outer membrane protein e of Escherichiu coli K-12 is co-regulated with alkaline phosphatase. J . Bacterial. 143, 151-157.