Vol. 171, No. 1, 1990 August 31, 1990

Cooperative Ildiko

mechanosensitive

Szsbo’,

Valeria CNR

Institute

of

July

Unit

for

the

SPA,

channels

Leonardo

in Escherichia

Guerra&

Physiology

Pathology,

General

13,

ion

Petronilli,

‘Glaso

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 280-286

BIOCHEMICAL

of

Fleming

2,

Mario

Zoratti”

Padova,

Italy

Mitochondria,

I’ia Trieste

Via

and

coli

75, 35121 Verona,

Italy

1990

A patch-clamp investigation was carried out on giant Escherichia coli stretch-induced as well as spheroplasts. The membrane exhibited “spontaneous” activity, with similar characteristics, i.e., a large number of conductance values arising from the cooperative behavior of channels in functional clusters. It appears likely that the same molecular species are responsible for both stretch-induced and “spontaneous” current conduction; the channel multiplexes can either respond to membrane stretch or function in an activated state, presumably brought about by the previous application of the mechanical stimulus. 01990 Academic Press, Inc.

The

recent

membranes

has

been

reported

application led

to

in of

KC1

Zoratti [5,6]

interesting

[3].

and

and

latter

and

still

channel rise

found

that

ii’]

exhibited

In the

bacterial

as well of

functional

studies membrane

a variety

as a about

clusters

141 detected in

has

coli

[2],

to

colleagues

fractions.

to

a conductance

stretch-sensitive,

membrane subtilis

[ll Escherichia

with

gives

Berrier

colleagues

Bacillus

mM KC11 channel

some

E.coli

technique

observations.

(350

The

gating.

purified

bacteria,

patch-clamp

a 1 nS

conductances,

containing faecsrlis

mM

cooperative

number

the

voltage-modulated

150

exhibiting

some

to possess

stretch-insensitive, 91pS

of

a large

proteoliposomes on

gram-positive

of

Streptococcus

of stretch-activated

conductances. In

this

communication,

spheroplasts.

It

conductance

actually

one

or

response stimulus

*To

whom

more

we arises

channel

types, stretch

they

become

correspondence

our

evidence

to membrane after

report

provides

from

the

patch-clamp that the cooperative

study 1 . nS gating

which may increase their or may function apparently activated.

should

ooo6-291x/90 $1.50 Copyright Q 1990 by Aca.femic Press, Inc. AlI rights of reproduction in any form reserved.

be addressed.

280

on

giant

stretch-activated in multiplexes

E.coli

of

open probability in without an applied

Vol.

171, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL

MATERIALS

AND

RESEARCH COMMUNICATIONS

METHODS

Giant spheroplasts (5-10 pm) were obtained from E.coli strain DHl by the penicillin method [81. After harvesting, the spheroplasts were suspended in 350mM KCl, 1OmM CaC12, 1mM MgC12, 5mM HEPES/KOH, pH 7.2. In all experiments reported the patch pipettes (Hildenberg 11411 glass, uncoated) were filled with the same medium. The data reported here were obtained from excised membrane patches. Experiments were conducted as described in [7]. Ohmic behavior was experimentally verified for most conductances (not shown).

RESULTS Voltage-clamped measurable of

spheroplast

step

about

9,

currents 24

application

of

invariably

resulted

(S-4)

and

40-58

in

al.

121,

the

it

A

is

more by

by

to

in

Fig. to

pores pS.

the

1A the

In

pipette

the

these

of

activated these

described

seemingly

1s

almost

under

phenomenon

operation

cases

interior

obtained

no

conductances

stretch

records is

either

with

large-conductance

typical

due

exhibited

channels)

mouth

of

presents

often

activity

or Hg)

activation

activity and

patches

sparse

(one cm

1 A-B

The et

only

(O-10

Fig.

circumstances. Martinac

or

suction

channels.

membrane

independent

1 2nS

*

0.5 0 1

L nS

B ;,

C

c

,‘I

1

#

.

1s -

A

LnS

A

Fig. 1. acti’vity. appearence (275 pS) induced point to pA (360 trace. (350 PS) and 107 separate

T.vpical stretch-induced (A and B) and spontaneous (C) channel A) V: +17 mV. Digitizing frequency: 6 KHz. Stretch induces the of 0.95-1.1 nS steps. The arrow ( 1 ) points to an approx. 4.6 pA Stretchevent. B) V: +87 mV. Digitizing frequency: 10 KHz. events. The largest steps fall in the range 1.2 - 1.3 nS. Arrows 36 pA (410 pS) (V), 60 f)4A (69: pS) ( v ), 87 pA (1 nS) ( 1 ) and 31 C) v: . Spontaneous activity. Continuous pS) (v) events. Digitizing frequency: 5 KHz. Arrows point to approximately 16.4 pA ( I), 48 PA (1020 PS) ( A ), 57 PA (1.2 nS) (v), 26 PA (550 PS) (8) This last transition cannot be resolved into pA (2.28 nS) (a) steps. steps even at higher resolution (40 KHz digitizing frequency).

281

by

Vol.

BIOCHEMICAL

171, No. 1, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS B

A

-

0.

0

c + 1 ns 4

v ---50ms

1 50 ms

2 ns 1

F

E

Fig. 2. fifembrane stretch-induced events. Records A-K were obtained from Digital sampling rate: 10 KHz unless otherwise specified. A) the same patch. V: -23 mV. Current steps: 7.4 pA (320 pS). B) V: -43 mV. Initial opening step: 22.5 pA (520 pS); fast closures: approx. 11.6 pA (270 pS). C) V: -33.5 Initial opening step: 32 pA (960 pS). Digital sampling: 20 KHz. IDV. Closures: approx. 16.4 pA (490 pS) ( 1 ), 22 pA (660 pS) ( v ). Notice the Digital sampling: 20 KHZ. Initial -33.5 mV. etepwise final closure. D) v: 27 pA (800 pS) (W), 24 opening step: 31.5 pA (940 pS). Closures (approx.): pA (720 ps) ( 1 ), 17.5 pA (520 pS) ( 7 ), 20 pA (610 1x8) (V). E) V: +67 mV. Digital sampling: 40 KHz. F) V: t27 mV, Digital sampling: 5 KHz. Current approx. 88 pA (3.3 steps: 8.6 pA (320 pS) ( 1 ); approx. 32 pA (1.15 nS) (‘I); nS) (V); 13 PA (480 pS) ( !).

approximately appeared

1 nS channels. instances,

experiment,

Fig.

1C

is

of

the

were

cycles Figs.

conductances

only

in

in Fig.

structure

of other

sizes

upon

at

permanent

a

stretch

involved of

of

to

from

the

of

of

their

activity point

of

general

experiment

but

give

outset formation.

The

the

suction,

activation

seal

case.

and

beginning

application

the

after

such

membrane the

from

applied in

conductances

anthologies

respectively.

sizes

observed most

comparative

catalogue

encountered

events Figs.

was

evident

the

repeated

stretch

same

behavior

the

1C.

involving

in

by

cases

3 present

stretch

conductance

their

the

was

obtained

elicited

of

active

suction

record

that other

2 and

membrane

to In

resulted

exemplified

shown

that

activity

no

typical

size

behavior.

channels

though

a

similar

both

kinetic

large-channel

even

presents

observed view

1B shows

frequently.

In other the

Fig.

and of the

is

They the

21):

records

are meant frequent

conductances

presented

measured

2C and

of

by

obtained to illustrate observation

Table

I.

The

1 nS.

Two

events

they

present

brief

partial

and

without

the variety of of a substate

themselves.

about

282

with

of

A more

complete

most

commonly

this

closures,

class due

are to

Vol.

171, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8

Fig. 3. Traces recorded without applying a transmembrane pressure gradient Digital sampling frequency: 10KHz. A) V: -23 mV. (spontaneous activit.v). Current steps: 12 pA (520 pS) (A) and 5.7 pA (250 pS) (~7). B) V: -41 mV. C) v: -63 mV. Major step sizes 47, 93 pA (1.15, 2.3 nS). Current levels: (from left): 306, 168, 136 pA (4.9, 2.7, 2.2 nS). Events at left and right were Notice the partial closures at separated by about 0.7 seconds of inactivity. Major steps: 80 pA (1.27 nS). E) Major opening: extreme right. D) V: -63 mV. The segment between the open triangles ( D d ) is shown 81 pA (1.2 nS). below with an expanded time scale (4X; digitized at 20 KHz). Arrows point to steps of about 22.6 pA (340 pS) (A); 14.8 pA (220 pS) ( A ); 19 PA (285 pS)(t).

transitions

to

functionally had a

a

conductance was

a

substates.

single

low probability

steady

channel:

of

pressure

events

several

opening

together

gradient

was

of the

closures

in

being

transitions.

For example, the 490 and 520 a match in the 480 pS transition

2D find

closures. possess

The a

transitions

In turn, 2F.

current

as

In

the

structure. closure

1 nS

transition. a closure

final

channels

at

four

due

4.4 to

the

2F, the

membrane.

of self-standing

closures

of Fig,

2F and rapid

doubt

that

1

of

in

Figs.

the

2C

approx.

half-amplitude

nS

conductancea

are

tale-telling

stepwise

2C.

into

by the spaced

Closure takes a series of approx. simultaneous

aggregates

events

but

nS.

283

have since

the

size

the

what

especially

partial

be organized

closely

step,

frequent,

Fig.

to would

across

with pS

attributed

shows

exemplified

Fig.

initial

Particularly in

very

approx. In

no

may

behavior,

former,

conduction by

leave

the

an

coincided

in turn,

substate

cooperative

single-step followed

the

2B, which,

observations

complex such

exhibiting and

of Fig.

event

be

channels

applied

the

520 pS

must

independent

Remarkably,

and

size

The

Bating

capable

of

in Fig.

2E

lead

to

shown

distinct

steps

place instead in a 1.1 nS openings is of

three

of

these

Vol.

171, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Conductance

values

I observed

consistently

in

a)

E.coli

spheroplasts

b)

+Stretch PS

cl

-Stretch

Substates PS

PS

155f30

150+35

140f25

240220

225225

220230

350225

350f30

330230

410230

425225

420+30

525225

550250

520f30

640230

650250

620230

760210

750flO

740230

850t25

850230

820+50

1000?60

1050+60

1250270

1200+60

The values are meant to be indicative of ranges of conductance values observed in symmetrical 350 mM KCl. Values below 120 pS have not been included. a) Conductance steps appearing upon application of stretch (suction) to otherwise silent patches, and disappearing upon release of suction. b) “spontaneously” from the beginning of the Conductances appearing experiment or persisting after repeated application of suction. cl Closureand-reopening transitions from “1 nS” stretch-induced single-channel events,

as exemplified

in Fig. 2C-D.

conductances. with

A

a fast The

spike

those

the

observed

while

also

Fig.

3C, extreme

opening

is followed In

the

membranes gating This

of

3B and almost exactly

channels In

together.

the In

nS

we

conductance

values.

independent

appears

again by

former, Fig.

sum of two

huge

to

be

observations two

3C, the consecutive

“1 nS” size

the such

channels

284

which

patches Fig.

no It

less seems

most

Fig.

than

occurred

see

3C). distinct that explanation.

presented

separately fast

the

cooperative

economical

open

2B;

12

Bather,

as those

shows

Fig.

unlikely

pores.

(Table

3A

1 nS channels is 3E : a one-step

(see also

of a 4.9 nS single,

steps

matched

(compare

closure

measured

closely

example, of the in Fig.

2D. stretch

steps

conductance

ended

membrane

inactive

as an

stepwise

so many

is supported

3C. the

1.5-5.5

single-step

concept

current

or

by Fig.

of

to otherwise

evident:

a 520 pS

began

as exemplified

The composite nature events such as the one

in

which

application

of the

stretch

by a protracted

possess

events

level, the

sizes

also

from

right).

range

“spontaneous”

The was

evident

observed

without

applying

pS closures

clearly

also

conductance

obtained

structure

250

again

to a high

same picture.

A substate

approx.

we

times

recordings

presented I).

few

event

a short

in but

Fig. close

matches time

later.

Vol.

171, No. 1, 1990

The

larger-amplitude

and

were

heavy

BIOCHEMICAL events

often

were

favored

characterized

flickering

(see

Fig.

AND BIOPHYSICAL

by

by

fast

RESEARCH COMMUNICATIONS

higher

transmembrane

kinetics,

sometimes

voltages

giving

rise

to

3D).

DISCUSSION The of

Results

both

the

match

the

stretch

the

units

operating

as

“clusters”

single,

have

particular, 2E-F

repeated

and

or

gate

even

structure

3B-Cj.

lengthy no

of the

The

size

of the

for

stretch-insensitive

pS.

authors

records.

The

insensitive” in the

current

this

report,

close

reported

behavior,

which

possible

structural

Which Madison

by way

conformational of the have

incompletely

[11,12]

to exist

can extend similarity the has

actually

from

to account

for

that

the

states

[ 101. as triplets

to clusters

channels

provided

On the

described evidence 285

SA channels.

At

closely

capable

porins

here

hand,

of

passing

and porins

E.coli

and

belong a

spaced

of E.coli

a pronounced These features

SA channel for

one

stretch-activated

exhibiting

the

the

conductances.

major

of triplets.

in their “stretch-

with

numerous,

other

medium.

the

coincide

different the

in the expected

our

that

are

to be

placed

in

“activated”

“modules”

with

et al. 121. value

[3]

to

modular

conductance

may

the

having

reported

between

al.

unit”

et al.

been

only

et

Delcour

a

be

of the

(See

capable

of

to be considered

SA channels not

half

In

including

become

by Martinac

of a “l/2

therefore

formed complexes,

clusters”

possibility

Delcour

occurrence

is to assume

membrane group

of

basic

factors,

tentatively

3E

current.

of

may

to one

Fig,

“clusters”

conduct

channel

might

substates, of

other

“clusters

mentioned

is close

arising

economical

porins

been

channel

described

to

self-

commonly

but

and

cases

contains

most

1 nS,

The

“module”

the

An identification generally

had

to

as those

ill-understood

applied.

range

ought

values

[9]:

channel

mentioned

in various

unlikely

extend the

is

possibility

most

conductance

form

This

activity

described present

also

conductance

110-130

Those

may

two

functional

The

about

I),

repeated

apparently

closures

give

the

(Table

by in the

such

to

of

on

cases

of the

of

channels.

may

two

membrane

combine

Depending

“1 nS”

size

bacterial

This,

induced

activity

sizes

conductance

stretch

interval the

the

membrane-stretch,

when

the

transitions

the

behavior

the be

and

structure

channels. in

that

with

stepwise

stoichiometries,

cooperative

Fig.s

variety

stretch-activated

a maximal

different

The

normally

of a composite

could

notion

model:

which

observed

the match

in

following

conductance

with

the

of regularity

favor

“spontaneous”

activity

support

conductances,

in

the

values

same channels.

a degree

been

and

“spontaneous”

strongly

to the

suggest

evidence

conductance

that

cycles

standing and

the

fact

due

presents

stretch-activated

between

and was

section

seems they have

cooperative point to a

porins. to is

localization

doubtful. on

the

The outer

Vol.

BIOCHEMICAL

171, No. 1, 1990

membrane

[9],

conductances found

in

to the is

that

inner

et

membrane bacteria

in

al.

[4]

have

fraction. like

gram-negative

assigned

Very

S. faecalis organisms

many

similar and

these

activity

B.

subtilis

channels

of

the

has

been

15-71.

reside

A

at

the

sites.

Inner

mitochodrial

1151 have

been

conductance well

Berrier

gram-positive

possibility contact

while

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

recently lrhe

to reflect

The

latter

[13,14]

been

channels.

be expected

function.

membranes

and

reported

endoplasmic

to host

similarity

of these

similarities

in their

remain

reticulum

similar, widely

membranes

sub&ate-rich

high-

distributed

strucuture

pores

as well

may

as in

their

for

useful

to be defined. Acknowledgment8

We warmly comments, Martinac

Prof. for

thank

Prof.

A. Peres

for

useful

publication,

and

thank

European

the

Mr.s

G.F. the

use

discussions Righetti Economic

Azzone

his

support

of some equipment,

and and

for

for

Santato

Community

providing for

technical

for

support

and

Dr.8 A. Ghazi manuscripts

“Stimulation-Science”. REFERENCES

Ill c21 c31 r41

r51 [61 [71

[81 [91 [lOI

[ill 1121

r131 [I41

r151

Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J. (1981) Pfluegers Arch. 391, 85-100. Martinac, B., Buechner, M., Delcour, A.H., Adler, J. and Kung, C. (1987) Proc. Natl. Acad. Sci. USA 84, 2297-2301. Delcour, A.H., Martinac, B., Adler, J. and Kung, C. (1989) J. Memb. Biol. 112, 267-275. Berrier, C., Coulombe, A., Houssin, C. and Ghazi, A. (1989) FEBS Lett., 259, 27-32. Zoratti, M. and Petronilli, V. (1988) FEBS Lett. 240, 105-109. Zoratti, M. and Petronilli, V. (1988) in “Molecular Basis of Biomembrane Transport”, Palmieri, F. and Quagliariello, E., eds. Elsevier. pp. 91-98. Zoratti, M., Petronilli, V. and Szabo’, I. (1990) Biochim. Biophys. Res. Comm. 168, 443-450. Kabach, H.R. (1971) Meth. Enzymol. 22, 99-120. Buechner, M., Delcour, A.H., Martinac, B., Adler, J., and Kung,C., (1990) Biochem. Biophys. Acta, 1024,111-121. Mauro, A., Blake, M. and Labarca, P. (1988) Proc.Natl. Acad. Sci. USA 85, 1071-1075. Schindler, H. and Rosenbusch, J.P. (1978) Proc. Natl. Acad. Sci. USA 75, 3751-3755. Schindler, H. and Rosenbusch, J.P. (1981) Proc. Natl. Acad. Sci. USA 78, 2302-2306. Petronilli, V., Szabo’, I. and Zoratti, M., (1989) FEBS Lett. 259, 137-143. Kinnally, K.W., Campo, M.L. and Tedeschi, H. (1989) J. Bioenerg. Biomemb. 21, 497-506. Simon, SM., Blobel, G., and Zimmerberg, J., (1989) Proc. Natl. Acad. Sci. USA 86, 6176-6180. 286

prior

assistance. under

and

the

B. to

We also program