Vol.

186,

July

31, 1992

No.

BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

PORIN

OF

PSEVLXIMONAS

AERVGUWSA

645-651

FORMSLOWCONDUCTANCE

ION CHANNELIN PLANAR LIPIDBILAYERS MAKOTOOBARAand TAIJI NAKAE

Departmentof Molecular Ljfe &Science Tokai University Schoolof Medicine,Isehara 259-11-Japan

Received June 1, 1992

SUMMARY Protein El, a porin of the outer membraneof P.seudomonns aeruginosn was reconstituted into planar lipid bilayers. Single channel conductance of the protein appeared to be 230 pS (pica siemens)in 1 M KU-10 mM Hepes,pH7.2. This value is approximately 5 times lower than the conductanceof the OmpFchannel of E.aYmri&iczmli. Conductanceincreasedlinearly as the membranepotential wasraised from -200 mVto +200 mV,and wasnearly proportional to the KC1concentration. Theseresults showthat protein El is probably a genuine poti in the P. ner~@nasnouter membrane supporting the earlier conclusionthat protein El formsa small channel. ‘D1992Academic mess,Inc.

The outer membrane of gram-negative bacteria constitutes poring. which

form

transmembranepermeability channels (1, 2). The size of the porin channels of enteric bacteria such as Eschertihia coli is close to that of saccharideswith Mr, SO0or diametersin the range of 1.1to 1.2 nm (1, 2). The tize of the porin channels of Pseuriomonns aauginess is a controversial issue. Protein F, reportedly a porin. forms a large polysaccharidepermeablebut extremely inefficient pore (3). Protein C, D2 and El (OprC, OprD, and OprE) form channels through which saccharideswith an Mr of approximately less than 350 to 400 can pass (4). The OprD channel showedseledivity toward basic aminoacids and imipenem and hence,wasthought to be a facilitated transporter (5). Recently a group of investigators reported that protein El (designatedas E2 in ret’. 3) had a wider channel than the

E.

mli

swelling method(3). To characterize the

OmpF channels as determined by the Liposome channel

by

a physico&emica.lly defined method

Vol.

186,

No.

2, 1992

that is basically

BIOCHEMICAL

different

AND

BIOPHYSICAL

the lipzzome swelling

from

OprE reconstitutti

into

RESEARCH

COMMUNICATIONS

technique, we determined the ion

conductivity

through

planar

Lipid bilayess.

The results

unequivocally

showed that OprE forms a lower conductance channel than the E. coli porins.

MATERIALS and METHODS OF PROTEIN El The outer membrane of P. apr~.@nosaPA01 was isolated and OprE was purified by DEAE-high performanceliquid chromatographyas reported (4). Purified OprE appeared to be homogeneous on a scdium dodecyl sulfate-acrylamide gel electrophoretogram(Fig. 1) and waskept in a solution containing 34 mMfl -cctylgluccside-I mMEDTA-10 mMTris, pH8.0at a protein colicentration of about i50 pg/ml. PLANAR EILAYERS AND CHANNEL INCORPORATZON Weformedbilayer membranes b,Ythe prcctiure describedby Montal and Mueller (6). A Teflon chamber, separating two aqueous phasesby a Teflon film (thicknm 0.25 pm) with an orifice of 0.3 mmin diameter, wasused throughout. Soy bean L-cr-phosphatidyl choline (Sigma T,ype U-S) or L- u-diphytanoyl phcsphatidyl choline (Avanti) was dissolved to the concentration of 10 mg/ml in hexane. Bilayer membranes were formed from two lipid monolayersat the air/water interphase by raising water level to an aperture of the Teflon septumpretreated with 1%hexadecanein hexane. Formation of the membranewas monitored by an increase of the eltctric;l capacitance te about 0.5 ~F/cmZ. Electrical resistance of the membrane was consistently more than 400 GQ/cmP. Purified protein was added to the cis compartment (in which a 10 Go probe was immersed) at a final concentration about 4 rig/ml, stirred by a magneticbar for 60 set and the conductivity increment was recordecj through Ag-AgC1 eltirodes connected to a patch clamp amplifier CEZ-2200 (Nihon Kohden) by the voltage-clamping mode. Membranepotential was applied through a stimulator SEN-3301 (Nihon Kohden). Signals were monitored by a digital recording mlmope Tektronix 2232 and a pen PURIFICATION

reco&~

LR4210

(Yokogawa).

HESULATS MACROSCOPIC

CONDUCTIVITYMEASUREMENTS

To test channel insertion into

bilayers.

macroscopicconductance increment upon addition of OprE was recorda at various concentrations of KU.

The conductance abruptly increased a 100 rig/ml of OprE was

addedto the 0.1 M KC1 bath. About a hundred-fold increaseof conductancewas achieved within 1 to 2 min (Fig. 2). The channel insertion rate was almast constant as the bath solution varied from 0.05 Mthrough 1 M KCI. SINGLE

CHANNEL

CONDUCTIVITY

MEASUREMENTS

Before running the single channel

experiments u&singOprE, we performedcontrol experiments to verify our technique using 646

Vol.

BIOCHEMICAL

186, No. 2, 1992 A

6

AND BIOPHYSICAL

C

co 0

* 200K -rl16K .

C-

0,

,.”

2 5 0

I

E-

01

100

: 5 F 0

-66K

**

10

-42K

I

02

_ .-

-

._--

RESEARCH COMMUNICATIONS

3_

I

1

I,,

3

,

I

I

5

10

15

TIME,

MIN

P&J Electrophoretic profile of purified OprE. OprE was purified from the isolated outer membrane of P. aeruginosa PA01 as d&bed earlier. Polyacrylamide gel (10%) electrophoresk was carried out according to the procedure described by Laemmli (14). Lane A: whole outer membrane protein. Lane B: purified OprE 2 &g, Lane C: molecular weight markers. Fig, Macroscopic conductance measurements. Lipid bilayers were formed from La-phcsphatidyl choline by the prccedure dmibed above, Approximately 100 rig/ml of purified Opt-E was added to the subphase of the bath solution, constantly stirred by a magnetic bar. The conductivity increment was recorded immediately. Applied membrane potential was +50 mV. (A) 1 M KCl, (B) 0.1 M KCl, (C) 0.05 M KQ. All bath solutions contained 10 mM Hepes. pH 7.2.

the well-characterized

OmpF porirl

of E. mli

R.

The single channel conductancesof the

OmpFchannel appearedto be 3.2 nS (nano siemens)in 1 M KCland the channeLscla& at the elevated membranepotential in a fashion of 3 dizrete steps with the conductanceabout l/3 of the above (data not shown). The results confirmed the previous results (7, 8). Channel activity

of gramitidin was also recorded and confirm& the report&

results

(9).

Thus the validity of our technique was confirmedby these findings, When4 ng per ml of OprE was addedinto the cis compartmentof the chamber,dkzrete single step conductance incrementswere observedwith cccasionalclcsing (Fig. 3). Unit conductanceincrement was fairly uniform. A histogramof single channel conductancesof 140channel even& showeda narrow distribution (Fig. 4). Average single channel conductancein 1 M KCl-10 mM Hem pH7.2 appearedto be 230 pS (pica siemens)at the applied membranepotential of +50 mV. 647

BIOCHEMICAL

Vol. 186, No. 2, 1992

i

~~~T~-~

1 ~ ,

_~-~

Fia. Stepwise conductance d&b& in the legend to Fig. 2 in Purified OprE (final concentration, octylgluctide concentration was les; showed no detmble influence on the

This result clearly

indicates

membrane of I? aeruginosa

that

of E. cdi

closes as the applied

1

~~

RESEARCH COMMUNICATIONS

g--cm--~

-+

increment by OprE. A lipid bilayer was formed as the bath solution of 1 M KCl-10 mM Hepes, pH 7.2. 4 rig/ml) in 0.2% octylglucazide was added. Final than 0.001% and this concentration of the surfactant conductivity. Applied potential was +50 mV.

the single

channel

conductance

of OprE of the outer

5 timessmaller than that of the E di

is about

Since some porin channels

AND BIOPHYSICAL

clase at a high membrane potential potential

OmpFchannel.

(e.g. the OmpF channel

exceeds 90 mV, ref. 8), we examined

the current

verws voltage relationship. Results of such experimentsshowedthat the OprE channel increased the currents

linearly

as the membrane potential was changed from -200 mV to

+200mV without indicating clear closing potentid (Fig. 5). To determine levels

the eff&T

of KC1 molarity.

of salt concentration,

The results

conductivity

shown in Fig. 6 indicated

648

was recorded that

at various

the average

single

Vol.

186,

No.

BIOCHEMICAL

2, 1992

40-

“‘.

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-

30-

:

i

iij

20-

;I: g n

io-

0

100

200

300

400

500

CONDUCTIVITY,

600

700

pS

m Histogram of conductance increments due to a single channel incorporation of OprE into bilayers bathed in 1 M KCl-10 mM Hepg. pH 7.2. Histogram represents 140 independent channd events similar to that shown in Fig. 3.

channel

condllctance

the bath solution, suggested

at 2 and 3 M KC1 were. after corrty3ton

by the specriric conductivity

about 80% of that in 0.1 M KC1 as seen in E. coli potin

that the channel

tzame

slightly

narrower

(8).

The result

at high ionic environment.

Although

0.1 0.3

0

6

of

1.0

2.0

3.0

KCIC0N~NTRATlON. M

Fig. 5. Current-voltage relationship of the OprE channel. Membrane was formed as described in the legend to Fig. 2 and 4 rig/ml of OprE was added. When several channels were incorporated and the channel activity became steady, applied membrane potential was fluctuated and currents were recorded. Conductance measurements with the membranes bathing in different of KCl. Experimental conditions were similar to that of Fig. 3 except bath solution used. Applied membrane potential was +50 mV. Symbols; 0 ,average single channel conductivity; 0,single channel activity normalized with the specific conductivity of the respective bath solution. Channel events were 45, 34, 140, 28 and 6 for 0.1, 0.3, 1.0, 2.0 and 3.0 M KU. respectively. Fin.

6

concentrations

649

Vol. 186, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the porin channels are generally less selective for small ions, there are examplff of porins that allow preferential diffusion of small inorganic ion(s) (10). Wetested the conductivity of the OprE channel for the permeability of small ions. Though the conductivity varied a little

among salts testecl, OprE allowed the diffusion of small ions without strict

mation

so far tested (data not shown).

DISCUSSION Wetested OprE, previously identified as a memberof the porin family of the outer membrane of P. eugim,

for channel-forming activity by determining the ion conductivity using

the planar membrane technique. The results showedthat OprE is the channel-forming protein characteristic of porins. This prorein showedthe single channel conductivity in 1 M KC1about 230 pS that is about 20%of the conductivity of the OmpFchannel of E. c&i. Wehave reported earlier that the outer membraneof P. wuginasn contains at Ipa% three channel proteins forming smaller-sizedchannels than the E. di

potins.

The results shown

in this study are fully consistent with our earlier conclusion. Although we could not calculate the precisepore diameter due to lack of information on the pore structure, it is paritive that the size of the channel of P. neruginasaOprE is less than that of the E. mli Q&.

Following on our report on the channel forming activity of OprC, OprD and OprE, a group of investigators reexaminedthe permeability

of the OprE by the l&some swelling

methudand reported that the size of OprE channel is larger than the E. c&i OmpFchannel (3). They criticized our data. claiming that the l&some swelling technique was not used under optimumconditions. Wehave reexaminedour technique carefully and concludedthat our experimental protocol wascorreX

Furthermore,we confirmed validity of our previous

results (11). In fact, the sameJiposomeswelling methodwas able to distinguish clearly a very slight difference in the maltodextrin selectivity of the wild type and mutant LamB 650

Vol.

186,

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

proteins (12). This small permeability difference among the LamB proteins was confirmed by the maltcdextrin technique investigators

binding to the proteins

it is difficult

by the planar membrane technique (13). With this

to cause artifacts

These arguments in turn suggest that the

who concluded that OprE forms a larger pore than the

E.

c&i OmpF should

reexamine their lipcsome swelling t&nique.

ACKNOWLEDGMENTS This study was supported by the grants Education of Japan, Culture and Science and the Naito Foundation. a Scholarship from the Ministry of Education of Japan.

from the Ministry of M.O. was supported by

REFERENCES 1. Nakae, T., (1986) CRC Crit. Rev. Microbial. 13, l-62 2. Benz, R. and Bauer, K.. (1988) Eur. J. Bitiem. 176, 1-19 3. Nikaido, H., Nikaido, K. and Harayama. S., (1991) J. Biol. Chem. 266, 770-770 4. Ymhihara, E. and Nakae, T., (1989) J. Biol. Chem. 264, 6279-6301 5. Trias, J. and Nikaido. H., (1990) J. Biol. Chem. 265, 15680-15684 8. Mont& M. and Mueller, P., (1972) Proc. Natl. Acad. Sci. USA, 69, 3561-3566 7. Lakey, J.M. and Pattus, F., (1989) Eur. J. Bicchem. 186, 303-308 8. Buehler, L.K. Kusumoto. S., Zhang. H. and Hcsenbuxh, J.P., (1991) J. Biol. Chem. 266, 24446-24450

9. Hladky, S.B. and Havdon. D.A., (1972) Bic&im. Biophys. Acta 274, 294-312 10. Hancock, R.E.W. and Benz. R., (1986) Bicchim. Biophys. Acta 860, 699-707 11. Nakae. T. and Ycshihara, E.. (1991) Antibiotics and Chemother. 44, 29-33 12. Nakae, T.. I&ii, J. and Ferenci, T., (1986) J. Biol. Chem. 261, 622-624 13. Benz, R., Francis G., Nakae, T. and Ferenci, T., (1992) Bia%m. Bia&ys. Acta 1104, 299307 14.

Laemmii. U.K., (1970) Nature 227, 680-685

651