Uncoupled Na+-efflux on reconstituted shark Na,K-ATPase is electrogenic

BIOCHEMICAL

Vol. 160, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 801-807

April 28, 1989

UNCOUPLED

Na+-EFFLUX

ON

RECONSTITUTED

SHARK

Na,K-ATPase

IS

ELECTROGENIC Flemming Institute

Cornelius

of Biophysics,

Received March

15,

University

of Aarhus,

Denmark

1989

In liposomes with reconstituted shark Na4K-ATPase produced to and ATP induce an contain sucrose addition of external Na uncoupled Na+ -efflux on inside-out oriented pumps which can be inhibited by digitoxigenin.This flux mode is found to be electrogenic and accompanied by hydrolysis of ATP. The coupling ratio of Nacyt transported per ATP split is 3:l measured as the initial rate of rise in transmembrane potential and initial rate of liberated Pi. 0 1989 Academic Press, Inc.

In

the

absence

catalyses

an

known

as the

eight

known In

from

uncoupled modes

the

with

no cations

the

ATP molecule red

free

extrusion transport

ghosts

solutions of

accompanied (s04--) of is

of

(6,7)

an El-form

(fig.l).

3:l

the

been

hitherto

(5).

Na+ i.e.

uncoupled

of the

This

extracellular

ratio

of

has

enzyme

suggests or

the

K+

3 Na+-ions

to

be

expelled

split.

cell

establishment

anions

a coupling

to

Na,K-ATPase

which

scheme

bound

of

the

be one

can support

a returning

absence

with

to

Albers-Post

an E2P-form

K+ (l-4)

and

Na-pump

accepted

or

SXtrUSiOn

Na+ -efflux

via

in

Na+

Na+

place

In

be

supported

widely

electrogenic

K+

extracellular

takes

transport

per

ATP

flux

the

Na+-efflux

of

loaded

of

Na+

a membrane by (8).

sodium electrically

an

with does

is

not,

potential

Ouabain

This

Na+ the

led

into

however,

lead

as expected

sensitive to

efflux

the

accompanied

efflux conclusion by

anions

and of

Na+ -and to

is

found

the

that and

the to

present

the that

active the

neutral.

801

0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 160, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

extracellular E,P#

E,P

tb

=

EIPNa

=

E1PNa2

=

E1PNa3 ti

‘i

L p(NaJ

E2

ATP E2A*

ADP E,A

=

E,ANa

=

ElANa

=

ElANa

cytoplasmic

Fig. 1. Simplified scheme for uncoupled Na+-efflux based on the Albers-Post model (6,7) as modified by Karlish et a1.(15) and Cornelius & Skou (9). The boxes signify enzyme species in rapid equilibrium. The scheme depict the two enzyme conformations El (the Na-form) and E2 (the K-form) and their phosphorylatedand occluded forms. Only EIA with 3 Na+ bound is assumed to be phosphorylated and be able to occlude Na'. In the

present

dogfish

Squalus

potential

as

Na+-efflux.

paper achantias

well The

intraVeSiCUlar

as

Na+

is

electrogenic

modes

to

interfere

restricted

to

very

apparatus

and

a

VI).

restricted vs.

to

time

With Na+ is 3

Na+:l

and

hence

and

to In

short

leads

order

uncoupled

the

build-up

of

to

the

exchange

avoid

membrane using

of which

two

potentials a

sensitive

within

transmembrane

exchange

intervals

were

stopped

fluorescent

dye

ATP-hydrolysis the

flow

rate

of

were hydrolysis

linearly. it

is

shown

by ATP-hydrolysis gives

to

spiny

Na+-Na+

measurements

increase

the

the

supported

of

potential

interval

from

accompanying

ATP

time

membrane

to

measure

flux-mode

measurements

preparation

accompanied

to

ATP-hydrolysis

(9).

a time

this

ATP

the

Likewise

was found

Na,K-ATPase

was used

uncoupled

which

(Oxonol

reconstituted

rise

with to

a

that

uncoupled

a stoichiometry

transmembrane

efflux close potential

of to as

predicted.

with incorporated shark In the experiments reported liposomes The fraction of enzyme molecules inserted Na,K-ATPase were used. as inside-out (i:o) was activated by addition of ATP. The determination of the orientation preparation of proteoliposomes, of inserted Na,K-ATPase and the recovery of specific hydrolytic activity was performed as previously described (10). 802

BIOCHEMICAL

Vol. 160, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The expression efflux and influx refer to the cellular situation and is therefore equivalent to uptake and extrusion the proteoliposomes due to the activation of exclusively oriented enzyme molecules.

in i:o-

With the time intervals used to complete the measurements of either hydrolysis or potential the increase in intravesicular Na+concentration was calculated to be less than 75 FM. The rate of ATP-hydrolysis on i:o oriented Na,K-ATPase was measured using [32P]ATP employing the method of Lindberg and Ernster (11). The membrane potential was assessed using the fluorochrome Oxonol VI essential as described by Ape11 et. al. (12) (see legend to fig.3). RESDLTSAlNDDISCUSSION In

fig.

2 the

time

proteoliposomes

ATP-hydrolysis

exposed

(n-o

oriented

ouabain.

As

indicated

the

whereas

of

preincubated

inhibit

within

course

ATP

with

enzyme

see ref.

10).

the

extended

test

periods

transported

Na+

is

stimulates

ATP-hydrolysis.

Taking

the

as representing

ATP-hydrolysis

is

of Pi-liberation

due

to

exchange

Na+-Na+ rate accompanying

also

of

sides

obtained:

only

constant, rates by

uncoupled

(t

Na+-efflux

[secl

Fig. 2. Rate of ATP-hydrolysis using proteoliposomes preincubated with ouabain (1 mM) in the presence of Mg2+ (1mM) and Pi (0.2 mM) in order to inhibit n-o oriented enzyme. Proteoliposomes were prepared in 260 mM sucrose, 2 mM MgC12, 30 mM Histidine @H 7.0). The test medium contained: Na' 104 mM, ATP 25 PM, Mg 1 mM histidine 30 mM and 32P-ATP at a specific activity of 7.7 10 15 cpm/mol. The dashed line corresponds to a specific activity of i/o-enzyme of 41.6 umol/mg.h. 803

by

< 15 s)

i Time

of

inward

accompanied

hydrolysis

to

contained

is

activation

order

both

increasing

yields

using

in

with

medium

relation

initial

depicted

and ouabain

The test

rate

probably

which

Pi

is

reconstituted

a curvelinear

15 seconds

This

Mg2+,

due to

first

hydrolysis.

hydrolysis

a

Vol. 160, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 3. Transmembrane potential generated by proteoliposomes engaged in Na+-efflux. uncoupled 100 )A1 of proteoliposomes produced in 260 mM sucrose, 2 mW MgC12 and 30 m&l Histidine (pH = 7.0) were added the cuvette containing 2.8 ml of an identical buffer with 530 nM Oxonol VI. The temperature was 23 OC. The fluorescence was measured using a Perkin-Elmer MPF 44A spectrofluorometer with excitation wavelength 580 nm (slit width 20 nm) and emission wavelength 660 nm (slit width 5 nm). When ATP is added an increased fluorescence (F) signifies the generation of a transmembrane potential, inside positive. The membrane potential at steady-state was estimated to be about 240 mV from calibration curves using different K+-gradients clamped with valinomycin to generate known Nernst' potentials (see ref. 12). Addition of 100 +I4 digitoxigenin to the cuvette caused the potential to decline with a time constant of about 420 s. The asterix indicates shift in sensitivity (3x). The inset demonstrate a stopped flow experiment showing the early fluorescence events accompanying the activation of uncoupled Na+-efflux. One syringe contained proteoliposomes in Na+ (130 Mg2+ (2 mM), histidine (30 m&I) and the other syringe d), . The oscilloscope contained the same medium and ATP (100 FM) recording depicts the initial 2.0 sec. when the fluorescence increases after mixing (the rapid downstroke of the trace). 804

BIOCHEMICAL

Vol. 160, No. 2, 1989

rate

of

42.0

cytoplasmic

+

4.1

Na+

is

corresponds

to

3.7

sites

nmoles

liposome of

3:l

per

is

15 FM in the

a K+

curve of

ca.

seconds

the

development what

of

containing

to

260

to the

indicating

membrane

potential

BY

calibration

of potentials

reached

addition development uncoupled Na+

of

at

constant

of

digitoxigenin the

Na+ -efflux electrogenic

due

to

the

the

and not Na+-Na+

longer

or anion give

rise

from

to of

symport to

fig.

in

from

the

medium

oxonol

which

VI

is

charge

(uptake

inhibits

of

the

exchange

the

to

membrane

potential

In

order

assure

(9)

K+-

membrane

be about

of

to

of

clamping

PM)

was calculated

the

ion-gradients.

by

(6.7

to activation

due

inside

collapse

transmembrane

the

proteoliposomes

Na+

signal

is

3 this

a disappearence

decline

15

to be expected.

digitoxigenin

valinomycin

805

than

to

was 420 s. observed

with

of positive

fluorescence

for

Na+ (13)

and leads the

of

the

potential

of

steady-state

an increase

with

fluorescence

addition

up

Comparing

ATP

transport

taken

of

As seen

membrane

using

time

of

the

per

diameter

should

presence

pump molecules

the

mV. The

a

The

oriented

potential

the

an inward

proteoliposomes).

equilibrium

in

using

s-l

mean

leaky

of

number

a stoichiometry

to

is

potential.

in

of

very

addition

increase

development

inside-out

the

if

the

Na+)

mM

5 Na+-pumps

test-times

Na+-pumping

mM sucrose

a fast

positive in

found:

+ 0.31

cytoplasmic

for

104

of Na+ are

(10). for

are

a transmembrane

is

nm

exchange

of

and

taking

mM cytoplasmic

onset

3.15

corresponds

200

proteoliposomes

of

45 molecules

activation

0.23

the

encountered

Na+ -Na+

6 mM an

(yielding

Unless

leads

of

be

at This

On average

Na+ -concentration to

n=lO)

pH=7.0).

(10)

This

liposome.

proteoliposomes

activation

is

the

(10).

that

SD,

OC,

inside-out means

+

number

mg protein

this

internal

(20

turnover

incorporated

into

(mean

obtained

per

assumed

second

wmol/mg*h

an initial

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

240 after

that

potential

was due

by increased

cytoplasmic

we also

used

the to

a stopped

Vol. 160, No. 2, 1989

flow

equipment

seen

from

in the

transmembrane i.e.

order

inset

less

in

than

potential be

calculated

calculated

from of

s)

stoichiometry per

8.8

+ 0.22

this

Na,K-ATPase ions and

are

blood

split

is

study

it

support

extruded

per.

10-l'

the

transport

contrast

(e.g.

electrogenic Since experiments the

enzyme

translocate are

currently

at

in

using

nm)

capacitance the

f

turnover

0.31

charges

s-l)

a

transported

in

with

However, to

in

(e.g.

Na+

is

806

Na+ red

results

in

the

with

Cl-)

nor at

to

antiport

development

reports

by Karlish

at

renal

pH

7.0

in

the

Na+-concentrations

at if

but

Na,K-ATPase.

calculated

assumed

this

pH = 8.5,

potential

experiments investigate

3 Na+

in

was found

contrast

is

which

Na+efflux

electroneutral

3:l

shark

indicating,

of

cytoplasmic 3

reconstituted

and

result

reconstituted of

progress

is

(200

with

uncoupled

rate

is

saturating

1).

the

Na+ -efflux

a stoichiometry

(fig.

A,

when extracellular

an anion

and

find

species

split

same

This

pH 8.5

using

of

This

hydrolysis

who

area,

Na+-efflux

potential

of

(14)

surface

that

electrogenic

symport

al.

membrane

= AC,(dV/dt)/ne

(3.15

is

neither

et.

in

v.

f SD (n=lO))

to

preparation,

significantly.

ATP

uncoupled

a transmembrane

decreased

indicated

proteoliposomes

concluded

of

both

the

obtained. is

H+).

increase

C. By comparing

(mean

establishment

however,

As of

scale

n = 5. The specific

ATP molecule

In

a cation

onset

time

The the

of

an

absent.

cells

of

pumps

f 0.10

can

K+ are

recording.

the

rate

s-l.

mean diameter

of 2.80

From

in

of

turnover

hydrolysis

ATP molecule

and

on the

rate the

inside-out

from

lag

encountered

cm = 1 CrF/cm2 and e = 1.6 calculated

no

initial

to

number

3

mixing

250 ms.

dt<15

the

rapid

fig.

the

(dV/dt,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

obtain

is

measuring

and the

to

potential

within BY

can

BIOCHEMICAL

to

occlude

non-saturating other

species

present only and Na+ with

BIOCHEMICAL

Vol. 160, No. 2, 1989

less

than

stoichiometry

3

Na+ of

less

are than

able

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

turn

over

as

well

giVing

a

3:l.

Professor J.C. Skou is acknowledged for helpful criticism . The financial support of The Danish Medical Research Council, Novo Foundation, and The Danish Biotechnology Centre for Biomembranes and the technical assistance of T. Feddema and H. Zakarias is gratefully acknowledged.

1. Garrahan, P.J & Glynn, I.M. (1967) J. Physiol. Lond. 192, 159174. 2. Garrahan, P.J. & Glynn, I.M.(1967) J. Physiol. Lond. 192, 175188. 3. Glynn, I.M. et al. (1974) Ann. N. Y. Acad. SCi. 242, 257-371. 4. Lew, V.L.,Hardy, M.A. & Ellory, J.C. (1976) Biochim. Biophys. Acta 323, 251-266. 5. Glynn, I.M. in: Progress in Clinical and Biological Research, vol 268A, pp. 435-460. (Skou, J.C., NBrby, J.G.,Maunsbach,A.B. & Esmann, M. eds.) Alan R. Liss, New York 1988. 6. Fahn, s., Koval, G.J. & Albers, R.W. (1966) J. Biol. Chem. 241, 1882-1889. 7. Post, R.L., Kume, S., Tobin, T., Orcutt, B. & Sen, A.K. (1969) J.Gen. Physiol. 54, 306s-326s. 8. Dissing, S. & Hoffman, J.F. In: Current Tpoics in Membranes and Transport, vol 19, pp. 693-695. (Hoffman, J.F. & Forbush, B, eds.) Academic Press, New York 1983. 9. Cornelius, F. & Skou, J.C. (1985) Biochim. Biophys. Acta 818, 211-221. lO.Cornelius, F. & Skou, J.C. (1984) Biochim. Biophys. Acta 772, 357-373. ll.Lindberg,O. & Ernster,L. (1956) Methods Biochem. Anal.3,122. 12.Apel1, H.-J. & Berth, B. (1987) Biochim. Biophys. Acta 903, 480-494. 13.Cornelius, F. & Skou, J,C. (1988) Biochim. Biophys. Acta 944, 223-232. 14.Karlish, S.J.D., Goldschleger, R., Shahak, Y. & Raphaeli, A. in: Progress in Clinical and Biological Research, vol 268A, (Skou, J.C., NBrby, J.G.,Maunsbach,A.B. & PP. 519-524. Esmann,M. eds.) Alan R. Liss, New York 1988. 15.Karlish, S.J.D., Yates, D.W., and Glynn, I.M. (1978) Biochim.Biophys. Acta 525, 252-264.

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