Interaction of Na,K-ATPase with artificial membranes. II. Expression of partial transport reactions

335

Biochimica et Btophyska Acta 832 (1985) 335-353 Elsevier

BBA 85283

Interaction of {Na^+ K"^)-ATPase with artificial membranes. II. Expression of partial transport reactions Béatrice M . Anner Deparlmenl of Phcirmacolog}-, Vniversily of Geneva Médical School. CMU. CH-1211 Oerieia 4 (Swiizerland) (Received April 29th, 1985)

Contents I.

Introduction

335

II.

Fluxes via dephosphoenzyme A. Passive Na"^ and K"^ fluxes B. Rb*-Rb"^ exchange mediated by |he dephosphoenzyme

335 335 338

III. Cation fluxes via phosphoenzyme A. K ^ - K \ R b - ' - R b * exchange B. Na"^-Na"*" exchange and uncoupled N a * transport C. Elecirogenicity associaled with Na"*"-K'*" exchange D. Chemical modification of active transport

339 339 341 342 343

IV. Leakage channel A. In liposomes B. In planar bilayers C. Models

347 347 348 349

Acknowledgements

349

Références

350

I. Introduction I n the preceding review [1] some physicochemical and structural aspects of the i n t e r a c t i o n of (Na"^ + )-ATPase w i t h artificial membranes were discussed. The second part of the review deals w i t h the functional expression of the reconstituted p u m p m o l é c u l e s . A s the g ê n e r a i features o f the reconstituted N a ^ - K ^ exchange process have been discussed i n previous reviews on ( N a ' + IC^j-ATPase (e.g.. Refs. 2 - 8 ) , the p r é s e n t review focuses on aspects that have n o t yet been discussed i n détail elsewhere. i.e. o n (i) the p a r t i a l

transport reactions expressed i n artificial m e m branes, ( i i ) the electrogenicity i n reconstituted Systems, ( i i i ) chemically m o d i f i e d transport and ( i v ) the leakage channel c o m p o n e n t o f the p u r i f i e d ( N a ^ + K ^ ) - A T P a s e m o l é c u l e uncovered i n a r t i f i cial membranes. I I . Fluxes via dephosphoenzyme liA.

Passive

Na

and K

fluxes

A r t i f i c i a l p h o s p h o l i p i d membranes are very p o o r l y p e r m é a b l e to cations. The flux across

0304-4157/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomédical Division)

336

the membrane o f sonicated egg PC hposomes is

l i p i d used t o f o r m the liposomes [17]. I f the typical

a r o u n d 8.5 • 1 0 " m o l / c m - per s at 3 7 ° C [9] and

passive

the Na"^ flux a r o u n d 1.7 • 1 0 " "* m o l / c m - per s at

" n o n s p e c i f i c leaks t h r o u g h the l i p i d d o m a i n " as

4 ° C [10]. S i m i l a r l y , protein-free egg PC liposomes

inferred b y K a r l i s h and Stein [18]. firstly,

prepared by the cholate-dialysis p r o c é d u r e display

s h o u l d be no relationship between the active and

Na *.K *

fluxes

described

above

were there

a comparable l o w p e r m e a b i l i t y for N a * , K * a n d

the passive transport p a t t e r n o f the enzyme and,

Rb^ o f about I - I O " '

m o l / c m - per s at 2 5 ° C

secondly, d i l u t i o n o f the l i p i d phase by extrinsic,

Reconstituted ( N a ^ + K"^ )-ATPase m o l é c u l e s i n

crease o f the Teak' density. I n o p p o s i t i o n to this

i m p e r m é a b l e l i p i d s should resuit i n a linear de-

[11]. the liposome membrane enhance the c a t i o n per-

p r é d i c t i o n , the passive N a * a n d

meability o f the liposome membrane, i.e., the K *

expressed o p t i m a l l y a l the same l i p i d / p r o t e i n r a t i o

fluxes

are

flux, by a factor o f about 100 a n d the N a * flux b y

that favors the active transport [12.19]. Further,

a factor o f about 40 [ 1 1 - 1 3 ] , T h i s results i n a

N a * a c t i v â t e s the rate o f the active and passive

typical ( N a * + K * ) - A T P a s e - i n d u c e d passive flux

N a * i n f l u x i n t o the liposomes w i t h s i m i l a r kinetics

pattern ( F i g . l A ) , the K * flux being

2-4-times

below 30 m M N a C I [19], i n d i c a l i n g that the pas-

fasler than the N a * fiux [14]. H i l d e n et al. [15]

sive N a * , K * fluxes are the expression o f the i o n o -

find i n c o r p o r a t i o n o f 0.32-0.39% external '^'^Na*

p h o r i c a c t i v i t y o f the p u m p m o l é c u l e [20] rather

w i t h i n 60 m i n at 2 5 ° C as compared t o about 1.5%

t h a n nonspecific leaks.

^^K

the same t i m e p e r i o d

A f u n c t i o n a l relationship between N a . K - p u m p

[16], y i e l d i n g a passive K * : N a * flux r a t i o close l o

incorporation within

a c t i v i t y and ouabain-insensitive. passive K 'leaks'

4

exists i n high potassium ( H K ) and l o w potassium

( F i g . l A ) . Conversely,

this

typical

passive

: N a * flux r a t i o is not observed i n cholate-di-

(LK)

sheep red cells where increased

Na*,K*-

alyzed egg PC liposomes c o n t a i n i n g dog k i d n e y

p u m p a c t i v i t y is accompanied

( N a * + K ^ ) - A T P a s e where the N a ^ flux is o n l y

p e r m e a b i l i t y . B o t h transport functions are associ-

slightiy slower than the K * flux [17]. S i m i l a r l y , i n

ated

soybean-PC liposomes reconstituted w i t h p i g k i d -

W h e t h e r the relationship between p u m p a c t i v i t y

ney (Na*-t: K * ) - A T P a s e by a

a n d passive c a t i o n fluxes i n ( N a * +

detergent-removal

with

the

M

and

b y increased

L surface

antigen

K* [21 ].

)-ATPase-

p r o c é d u r e , the passive N a * and R b * i n f l u x rate is

liposomes is related to the association observed i n

equal

L K / H K sheep red cells is not yet k n o w n .

[18].

specific

Perhaps

cation

the

( N a * + K*)-ATPase-

permeability ratio

is

'short-cir-

c u i t e d ' by a relatively high p e r m e a b i l i t y o f the

The

cation-permeability induced

by

( N a ' -I-

K * ) - A T P a s e i n liposomes has n o t yet been fully characterized. P r e l i m i n a r y d é t e r m i n a t i o n s indicate

K, M g , N a

Rb

that, for example, c h o l i n e and phosphate ions can f l o w t h r o u g h the pores, whereas sulfate ions cannot ( A n n e r and M o o s m a y e r . u n p u b l i s h e d data) i n contrast

to

liposomes

reconstituted

with

Ca"*-

ATPase, w h i c h become p e r m é a b l e aIso t o sulfate ions [22]. A t y p i c a l passive flux p a t t e r n has

also

been described for hog gastric ( H * - t - K * ) - A T P a s e [23]. I t is p r o b a b l y p e r t i n e n t to the nature o f the A.

2-A

1

B,

Fig. 1. Passive Na^. K * or Rb"* fluxes via dephosphoenzyme. Fig. l A shows Na*.K"^-fluxes occurring in the simultaneous présence of internai and external Na*. K * or R b * and Mg^* ions as described in subsection IIA. Fig. I B shows the ouabainand vanadate-sensitive Rb* fluxes observed in the absence of external Na ' and Mg^* as described in section I I B of the présent review.

( N a * - t - K * ) - A T P a s e - i n d u c e d passive fluxes

that,

i n g ê n e r a i , the l i p i d c o m p o s i t i o n o f the membrane plays a crucial r ô l e i n the f o r m a t i o n o f such leaks. G l y c o p h o r i n . for instance, induces very l i t t l e perm e a b i h t y i n pure egg PC liposomes [24]. However, w h e n the l i p i d phase contains, for example, d i o l e o y l P C or other unsaturated PCs, g l y c o p h o r i n o r band

3 p r o t e i n induce

a fast l i p i d - t r a n s b i l a y e r

337

movement and i n t u r n a high p e r m e a b i l i t y to K * ions and glucose [25,26], I n vesicles formed w i t h the original erythrocyte lipids, g l y c o p h o r i n p r o duces no permeability increase [26]. Besides fatty acid saturation, p h o s p h o l i p i d headgroups such as serine and ethanolamine, as well as the p r é s e n c e o f c h o l e s t é r o l , play a crucial r ô l e i n sealing the p r o t e i n / l i p i d interface [27]. Effects o f l i p i d o n m e m brane permeability are also reported i n m a m m a l i a n red b l o o d cell m e m b r a n e s , where ouabain-sensitive and ouabain-insensitive K * transport, as well as K * , chloride and phosphate permeabilities augment i n a correlated fashion w i t h increasing PC content o f the membranes and decrease w i t h increasing s p h i n g o m y e l i n content [28]. Increasing the p r o p o r t i o n of unsaturated fatty acids [29] as well as membrane l i p i d p e r o x i d a t i o n [30] can produce increased K * flux or even K * leaks i n h u m a n red cell membranes, w h i c h i l l u s t r â t e s that also i n native membranes, spécifie lipids are required to keep up their barrier function. M a n y différent mechanisms can be envisaged for the permeability induced by p r o t e i n - l i p i d interaction (e.g., Réf. 31). Here we indicate only a few examples o f p r o t e i n effects on l i p i d bilayer properlies. Besides the enhanced transbilayer movement of PC mentioned i n the previous paragraph, faults i n l i p i d packing (e..g. Réf. 32), effects on l i p i d fluidity (e.g., Refs. 33,34) and t r a n s i t i o n t e m p é r ature (e.g.. Réf. 35), or local, p r o t e i n - i n d u c e d membrane t h i n n i n g or t h i c k e n i n g [36] have been evoked. Besides proteins, d é t e r g e n t s can increase l i p i d bilayer permeability. O c t y l glycoside at 25 m o l % , for instance, induces cation p e r m e a b i h t y b u t l i t t l e anion permeability [37j. W i t h regard to the ( N a * + K * ) - A T P a s e - i n d u c e d permeability, residual d é tergent effects o n the passive fluxes have been excluded o n the g r o u n d of two lines o f é v i d e n c e : (i) cholate-dialyzed protein-free liposomes do not display the typical K * : N a * flux r a t i o [11] and ( i i ) the passive N a * , K * - p e r m e a b i l i t y is relatively i n sensitive to the extent of detergent-removal ( A n n e r and Moosmayer, unpublished data). The p r é s e n c e o f proteins or d é t e r g e n t are not necessary to create spécifie membrane p e r m e a b i l i ties. For instance, a p e r m e a b i l i t y sélective for K * and non-electrolytes such as glycerol and e r y t h r i -

t o l appears i n pure egg PC liposomes w i t h i n creasing fatty acid u n s a t u r a t i o n and is decreased i n p r o p o r t i o n to the c h o l e s t é r o l content [38,39], to cite o n l y a few examples. I t has even be proposed that p h o s p h o l i p i d s are the actuai ion-carriers i n membrane transport Systems [40]. The f o r m a t i o n o f ion-channels i n the absence o f p r o t e i n can be imagined o n the basis o f the mixed-chain-length m o d e l [41]. W i t h respect to the ( N a * - l - K * ) ATPase-induced p e r m e a b i l i t y such strictly lipid-reiated effects can be excluded as the passive K * a n d N a * fluxes are related to the active transport p a t t e r n of the reconstituted enzyme and not to the p r é s e n c e o f enzyme lipids. T h i s is documented. for example, by the l o w K * p e r m e a b i l i t y o f liposomes reconstituted w i t h detergent-denatured enzyme, a s i t u a t i o n where the solubilized ( N a * + K. ' )ATPase hpids have f u l l o p p o r t u n i t y to enter the l i p i d phase under the e x p é r i m e n t a l conditions used [42]. Thus, w i t h regard to the passive fluxes i n duced by the ( N a * - l - K * ) - A T P a s e i n liposomes, the lipids seem to be i n v o l v e d i n d i r e c t i y i n m a i n t a i n i n g a transport-active p r o t e i n c o n f o r m a t i o n at a critical U p i d / p r o t e i n r a t i o . I n conclusion, the passive N a * , Ï C * - f l u x e s appearing i n the reconstituted ( N a * - I - K ' )-ATPase System are apparently the expression o f the enzyme's i o n o p h o r i c a c t i v i t y . Consequently. t h è s e transmembranous fluxes are the vectorial expression o f the unreconstituted enzyme's c a t i o n a f f i n i t y . A s a matter o f fact, the a f f i n i t y of the unphosp h o r y l a t e d ( N a " + K * ) - A T P a s e for K * is 2 - 3 - f o l d higher t h a n for N a * , as Skou has demonstrated b y c o m p a r i n g the effect o f N a * or K * o n the reactivi t y o f the enzyme towards yv-ethylmaleimide [43.44]. T h e excellent correspondence between the N a * : K * a f f i n i t y r a t i o o f the isolated enzyme and the N a * : K * flux r a t i o o f the enzyme after incorp o r a t i o n i n t o hposomes ( F i g . l A ) is another s t r o n g argument for a speciflc r ô l e of the u n p h o s p h o r y lated p u m p m o l é c u l e s i n m e d i a t i n g passive N a * and K * fluxes. T h u s . the ( N a * 4 - K * ) - A T P a s e may c o n t r i b u t e to the high p e r m e a b i l i t y o f n a t u r a l membranes. I t is w e l l k n o w n that n a t u r a l membranes and i n finitely m o r e p e r m é a b l e to K * ions [45] t h a n are, for instance, pure PC membranes [10]. The fundam e n t a l l y d i f f é r e n t nature o f biological and a r t i f i cial protein-free membranes is illustrated by their

338

distinct reactivity towards a m p h i p h a t i c m o l é c u l e s . The p a r t i t i o n coefficient of chiorpromazine, for instance, is about 15 000-times lower i n e r y t h r o cyte ghosts as compared to liposomes [46], w h i c h led the authors to suggest that biological m e m branes have a m u c h larger " i n t e r n a i pressure" than hpid bilayers, presumably because o f longrange ordering effects o f the membrane proteins on l i p i d fatty acid chains. The p r o f o u n d effects of proleins on membrane p e r m e a b i l i t y and i n t e r n a i pressure is certainly a planned and carefully organized characteristic o f biological membranes, i f this were not so. membrane-related processes w o u l d be chaotic and i n c o n s é q u e n c e cell organization, c o m m u n i c a t i o n and survival w o u l d be impossible. The characteristic K * and N a * permeability induced by ( N a * - I - K * ) - A T P a s e m o l é cules may be the expression o f such a b u i l t - i n bilayer-modifying f u n c t i o n . M e m b r a n e fusion, for instance, is deeply infiuenced b y the physicai and Chemical properties o f the membranes i n v o l v e d . Thus. i n fusion-mediated processes as intracellular membrane traffic and endo- or exocytosis [47], the p r é s e n c e and density of ATPase m o l é c u l e s - by their bilayer-modifying properties - may participale i n m o d u l a t i n g cellular membrane flow, IIB. Rh^-Rb^ phoenzyme

exchange

mediated

by the

dephos-

N o t only is the a f f i n i t y o f the unphosphorylated ( N a * + K * } - A T P a s e higher for K.* than for N a * ions (see preceding section), b u t the enzyme actually 'occludes' K * or R b * ions [ 4 8 - 5 0 ] . I n c o n s é quence, a or R b " flux occurring i n the absence of A T P is presumably the vectorial expression o f dephosphoenzyme m o l é c u l e s exchanging 'occluded' K * ions w i t h the surroundings. E x p é r i m e n t a l é v i d e n c e for dephosphoenzymemediated R b * fluxes has been presented by K a r l i s h and Stein [51]. I n the absence o f N a * and M g ^ * . vanadate- and ouabain-sensitive R b * fluxes ( F i g . I B ) appear i n the ( N a * + K * ) - A T P a s e - i i p o s o m e s [51]. N a * and M g ^ * i n h i b i t the process. A t external R b * concentrations below 200 / i i M , the concentration for h a l f - i n h i b i t i o n o f the vanadate-sensitive R b * flux is a r o u n d 1 m M for N a * and close to 1.3 m M for M g ^ * [51,52]. Conversely.

the

typical ( N a * - i - K * ) - A T P a s e -

K, Rb^Mg

Na,Mg

A, Fig. 2. (A) K * - K * or R b * - R b * exchange via phosphoenzyme as described in subsection IIIA of the présent review. (B) N a * - N a * exchange via phosphoenzyme as described in section I I I B of the présent review. Po.ssible couphng ratios of influx to efflux are indicated.

induced passive N a * and K * fluxes reviewed i n the preceding section were m o n i t o r e d i n active transport c o n d i t i o n s , e.g., i n the p r é s e n c e of 5 m M Mg2* plus 50 m M N a * and K * ( F i g . l A ) . I n c o n s é q u e n c e , the vanadate- and ouabain-sensitive R b * fluxes were i n h i b i t e d presumably by ( i ) h i g h a f f i n i t y N a * b i n d i n g to a cytoplasmic site, ( i i ) l o w - a f f i n i t y N a * b i n d i n g {K^ o f about 8 m M ) to an extracellular b i n d i n g site on the p u m p m o l é c u l e [51,52], and b y ( i i i ) o c c u p a t i o n o f a cytoplasmic site b y M g ^ * ions [51,52]. T h u s , the enzyme-characteristic passive N a * and K * fluxes described i n the preceding section are d i f f é r e n t f r o m the vanadate- and ouabain-sensitive R b * exchange process mediated b y the N a * - and M g ^ * - f r e e dephosphoenzyme reported by K a r l i s h and Stein [51,52]. T h e rate o f **^Rb* uptake is greatly enhanced b y the p r é s e n c e o f 20 m M R b C I i n the N a * - and M g ^ ^ - f r e e liposomes as c o m p a r e d to the uptake rate b y Rb*-free hposomes, suggesting an R b * R b * exchange process [51]. T h a t this exchange pro('ess is m e d i a t e d b y the i o n - p u m p m a c h i n e r y o f the ( N a * - I - K * ) - A T P a s e m o l é c u l e is demonstrated by (i) the b l o c k o f a saturable R b * flux fraction b y ouabain-pretreatment, ( i i ) the 50% decrease i n the R b * uptake by external vanadate ( b l o c k i n g the 50% inside-out p u m p m o l é c u l e s ) and ( i i i ) the almost 100% i n h i b i t i o n b y the simultaneous p r é s ence o f i n t e r n a i as well as external vanadate. R b * half-saturates the cytoplasmic b i n d i n g sites at a c o n c e n t r a t i o n o f about 0.6 m M , a n d the extracellular sites at a b o u t 0.2 m M , i.e., the R b * a f f i n i t y o f the i n t e r n a i and external b i n d i n g sites

339

of the dephosphoenzyme is not m u c h d i f f é r e n t , i n

u p to 30-times faster than the rate o f the dephos-

contrast to the s i t u a t i o n i n the

phoenzyme-hnked

phosphoenzyme,

R b *-Rb * exchange

and

pré-

where the R b * or K * affinity o f the i n t r a c e l l u l a r

sents then r o u g h l y a f i f t h

o f the A T P - i n d u c e d

cation sites is lowered by several orders o f m a g n i -

N a * - R b * exchange rate [54].

I t has been proposed

tude compared to the extracellular site [4-6,43,44],

that, like K * , M g ^ * may be b o u n d and released

The rate o f the vanadate-sensitive

d u r i n g the enzyme turnover cycle [55]. The s t i m u -

R b * - R b * exis a l -

l a t i n g effect o f M g ^ * o n the exchange rate ex-

most 200-fold lower than the rate o f the A T P - d e -

p l a i n s w h y H i l d e n and H o k i n [16] observed o n l y a

change mediated b y the dephosphoenzyme pendent

N a * - R b * exchange

observed

i n active

3-fold s t i m u l a t i o n b y ( A T P + P, ) o f the basai

K*

transport conditions [51]. Whether such l o w - r a t e

i n f l u x rate at 100 m M i n t e r n a i a n d external i n the

cation fluxes have a physiological significance and

absence o f M g ^ * ,

whether they could become

10 m M i n t e r n a i a n d external R b C l plus 5 m M

more i m p o r t a n t i n

vivo by an a m p l i f y i n g mechanism is not yet l i n o w n ,

i.e., the same effect as seen w i t h

M g C l , [54].

Regardless o f the possible function o f the R b *

T h e phospholigands orient the release o f the

slippage, its characterization is helpfui for better

occluded K * by c h a n g i n g the b i n d i n g a f f i n i t y o f

understanding the effect o f phosphoHgands o n the

extra- and i n t r a c e l l u l a r K * b i n d i n g a n d changing

p u m p function.

the c o n f o r m a t i o n o f the w h o l e p r o t e i n , p r o v i d i n g access o n l y

I I I . Cation fluxes via phosphoenzyme

t i o n s [57], IIIA.

K *-/: *, Rb^-Rh*

from

one

side

o f the

membrane.

A c c o r d i n g to models [56] or theoretical c o n s i d é r a by

exchange

the i o n m o v e m e n t must be accompanied

the b i n d i n g site itself or i m p l y an é q u i v a l e n t

reaction. A c c o r d i n g to T a n f o r d [57], the r e t u r n to Mg2*-free (Na*-K K * ) - A T P a s e

the o r i g i n a l c o n f o r m a t i o n . E , , must occur w i t h an

liposomes prepared i n 100 m M K C l / 3 0 m M i m -

unoccupied b i n d i n g site, W h e t h e r this theorem is

In

N a * - and

i d a z o l e / 1 m M E D T A , the simultaneous a d d i t i o n

c o m p a t i b l e w i t h the proposai that, under p h y s i o -

of 5 m M A T P and 5 m M P| increases the rate o f

logical c o n d i t i o n s , A T P a c c é l é r â t e s the conversion

• * ^ K * uptake by a factor o f about 3 [16]. whereas

of

ATP

E , K w i t h s u b s é q u e n t release o f K * at i n t r a c e l l u l a r

or P| added separately has no effect. S i m i -

larly, i n ( N a "

K * )-ATPase liposomes c o n t a i n i n g

K * sites) to

l o w - a f f i n i t y K * sites [58,59] remains to be studied.

10 m M R b C l and 5 m M M g C l 2 , o n l y the simulta-

An

increase i n the A T P or P, concentrations

and 3 m M

b e y o n d their o p t i m a l a c t i v a t i n g level progressively

s t i m u l â t e s ( F i g . 2 A ) the **^Rb* uptake rate

i n h i b i t s the K * - K * exchange process [54] by freez-

neous a d d i t i o n o f 5 m M phosphate ATP

E T K (extracellular h i g h - a f f i n i t y

about 3-fold [51,5]. Ouabain i n h i b i t s the i n i t i a l

ing

^*'Rb * influx when i t is b o u n d to the receptor site

f o r m w h i c h leases K * at the i n t r a c e l l u l a r side or i n

the System either i n the A T P - i n d u c e d

Ei(K)

located w i t h i n the liposomes, i n d i c a t i n g t h a t a

the P - i n d u c e d E 2 ( K )

pump-mediated K * - K * exchange process, such as

the extracellular side. T h e same pattern is seen i n

that described for red b l o o d cell ghosts [53], is at

red

f o r m w h i c h releases K " ^ at

cell ghosts [60,61].

w o r k . External ouabain has no effect o n the ( A T P

The K * - o c c l u s i o n step is a precursor reaction'

-t- P, )-stimulated "^Rb uptake [52,54], whereas ex-

and is not d i r e c t l y c o u p l e d to the E ^ - E j t r a n s i t i o n .

ternal vanadate at 18 j u M concentration blocks the

R a d i a t i o n i n a c t i v a t i o n o f ( N a * - f K * ) - A T P a s e re-

exchange process [52].

veals a target size for the K * - o c c l u d i n g mechanism

The rate o f K * or R b * exchange is stimulated by

increasing R b C l concentrations inside or out-

of o n l y 3 9 - 6 0 k D a as compared to the target size of

140-180

kDa

for

side the vesicles and becomes o p t i m a l above 150

nitrophenylphosphatase

mM

kDa

internai and 16 m M external R b C l . F u r t h e r ,

the

K *-stimulated

p-

activity and o f 1 9 0 - 3 3 0

for the ( N a * + K * ) - A T P a s e a c t i v i t y [61,62],

the p r é s e n c e o f 5 m M MgCl2 s t i m u l â t e s the ( A T P

suggesting

+ P| )-activated R b * - R b * exchange up to 10-fold

preserved

[54],

exchange

phatase activities are destroyed. I n agreement w i t h

process mediated by the phosphoenzyme becomes

this s t r u c t u r a l study, Jorgensen a n d Petersen [63]

so that the rate o f the R b * - R b *

that the K * - o c c l u d i n g mechanism even

though

the ATPase

and

is

phos-

340

show that b l o c k i n g o f sulfhydryl groups reversibly

p u m p increases for N a * ions a n d decreases for

abohshes the E j - E j t r a n s i t i o n , w h i l e the K * - o c c l u -

ions w i t h increasing p H , i.e., the p r o t o n a t e d p u m p

sion capacity remains intact.

has a h i g h K * - a f f i n i t y and the d e p r o t o n a t e d p u m p

The fact that, i n contrast to the K. ^-occlusion

a h i g h N a * a f f i n i t y . A T P has a ' d e p r o t o n a t i n g '

capacity o f the ( N a * + IC*)-ATPase, the K * - K +

effect, i,e,, i t favors N a * b i n d i n g to the enzyme

exchange reaction [16,51,52.54] as well as the E1-E2

[71,72], A t the same time, A T P antagonizes the

transition [63,64] are sensitive to i n h i b i t i o n o f the

d e p r o t o n a t i o n i n d u c e d b y the b o u n d N a * , w h i c h

ATPase

activity, i l l u s t r â t e s that

the

transmem-

means that

the p h o s p h o r y l a t e d

enzyme

has

an

branous i o n movement requires c o u p l i n g o f the

increased tendency to become p r o t o n a t e d , i.e., to

occlusion step to the E^-E^ t r a n s i t i o n . Detailed

b i n d K * ions and release N a * ions [71]. Thus, the

kinetic aspects and possible reaction schemes o f

p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n step as well as

the K * - K * exchange via K * occlusion are dis-

the p H at the i n t e r n a i and external

cussed and reviewed elsewhere [18,65].

surface

Structural studies using s é l e c t i v e proteolysis o f the a-subunit, c o n f i r m that the basic c o n f o r m a -

can

regulate

membrane

the relative N a * a n d

K*

a f f i n i t y and presumably also the N a * - K * transp o r t r a t i o o f the enzyme.

tional change o f the (Na*-)- K * ) - A T P a s e , the E , -

The a d d i t i o n o f the p r o t o n a t i o n - d e p r o t o n a t i o n

E2 transition [ 3 - 6 , 6 6 ] is necessary for the ( A T P -l-

reaction to the previous i n f o r m a t i o n leads to the

P|)-induced K * - K * exchange r é a c t i o n . Jergensen

f o l l o w i n g scheme ( T a b l e I ) : b i n d i n g o f N a ions to

et al. [64] pretreated

the ( N a * - I - K * ) - A T P a s e m o l é c u l e (or d e p r o t o n a -

ATPase

in

the p u r i f i e d

well-defined

(Na*+K*)-

proteolytic

conditions

t i o n or A T P b i n d i n g leading to N a * b i n d i n g )

at

favors the deprotonated, relaxed E , c o n f o r m a t i o n

spécifie locations. A split near the a m i n o t e r m i n a l

associated w i t h p r é d o m i n a n t a-helix content. Pro-

known

to cleave

the

a-subunit

polypeptide

of the p o l y p e p t i d e blocks the E^-E2 t r a n s i t i o n as

t o n a t i o n induces K * b i n d i n g ( o r K * b i n d i n g or

well as the R b - R b exchange process. I n c o n s é -

d e p h o s p h o r y l a t i o n induces p r o t o n a t i o n ? ) , transi-

quence, the R b - R b exchange seems to be t i g h t l y

t i o n to the tense E j c o n f o r m a t i o n associated w i t h

coupled to the E 1 P - E 2 P t r a n s i t i o n [64]. Such basic

h i g h sait b r i d g e and hydrogen b o n d c o n c e n t r a t i o n ,

c o n f o r m a t i o n a l changes seem to be associated i n

and

g ê n e r a i w i t h vectorial c a t i o n movements mediated

regard to the q u a r t e r n a r y p r o t e i n structure o f the

by membrane ATPases [67], Gresalfi and Wallace

(Na*

[68]

stabilize ^-sheets, and d e p r o t o n a t i o n , a-helixes.

demonstrate,

using circular d i c h r o i s m spec-

troscopy, that the E , - E 2 t r a n s i t i o n corresponds to

prédominant

^-sheet structure. T h u s .

+ K*)-ATPase,

protonation

with

appears

to

Protons seem to favor f o r m a t i o n o f a salt-bridge

an extensive c o n f o r m a t i o n a l change o f the peptide

between

backbone,

negatively charged c a r b o x y l groups and, according

presumably

involving

transformation

positively charged

amino

groups

and

f r o m an a-helix to a ^-sheet structure. Such r a p i d oscillations o f the quaternary ( N a * -I- K * ) - A T P a s e structure requires p r o m p t and r é versible rearrangement

w i t h i n the peptide

back-

bone, presumably b y m i n i m a l but critical m o d i f i cations i n the i o n i z a t i o n o f c a r b o x y l and

TABLE I CHEMICAL AND STRUCTURAL CHARACTERISTICS OF THE E , AND T H E E ^ CONFORMATION O F T H E (Na* + K " )-ATPase

amino

groups. The changed i o n i z a t i o n c o u l d r a p i d l y and reversibly m o d i f y the sulfur, hydrogen and

sait

bridges between d i f f é r e n t peptide segments. Protons are the obvions candidates for such a regulatory rôle, as Skou has proposed [69] and p r o v e n [ 7 0 - 7 2 ] . The enzyme is p r o t o n a t e d w h e n K ' ions are b o u n d and deprotonated w h e n N a * ions are b o u n d , i.e., K * b i n d i n g leads to p r o t o n uptake b y the (Na*-I- K * ) - A T P a s e and N a * - b i n d i n g to p r o ton

release [70,72]. I n t u r n , the a f f i n i t y

of

the

E,

E2

Chemical characteristics Cation bound Protonation Hydrogen bond content Sait bridge content

Na' deprotonated low low

K* protonated high high

Structural characteristics Physicai state a-hehx content ^-sheet content

' relaxed' high low

' tense' low high

341

to above scheme, transition from p r é d o m i n a n t ahehx (relaxed?) to p r é d o m i n a n t ;S-sheet structure (tense?). I n summary, c o n c o m i t a n t (i) phosphorylation-dephosphorylation, ( i i ) d e p r o t o n a t i o n - p r o tonation, ( i i i ) N a * , K * - b i n d i n g , ( i v ) E ^ E j transition, (v) relaxed-tense c o n f o r m a t i o n , ( v i ) low-saltbridge-high-salt-bridge content, and ( v i i ) a-helix^-sheet transition occur d u r i n g ( N a * + K * ) ATPase-mediated cation exchange. A s the E[-E2 t r a n s i t i o n is also i n v o k e d for the partial K*-K* exchange reaction, the p r o t o n a t i o n - d e p r o t o n a t i o n mechanism must also regulate K * uptake and release. Enzyme forms w i t h high K * affinity must be highiy p r o t o n a t e d and vice versa. T h i s p r i n c i p l e a g r é e s w i t h the fact that the unphosphorylated f o r m is rather p r o t o nated, has a high K * affinity and 'occludes' K * . As the b o u n d K * ions favor p r o t o n a t i o n , the enzyme goes over i n t o a stable E j K f o r m . The a d d i t i o n of ' d e p r o t o n a t i n g ' phospholigands ( A T P + P|) produces K * release, c o m p l e t i o n of the K * uptake-release cycle, and so increase of the K ^ - K * exchange rate. IlIB.Na-Na

exchange

and uncoupled

Na

transport

I n the absence o f K * , the ( N a * - H K * ) - A T P a s e from Squalus acanthias, p u r i f i e d and reconstituted in liposomes [15], performs o u a b a i n - i n h i b i t a b l e N a * - N a * exchange i n the p r é s e n c e fo 120 m M N a C I / 5 m M M g C l 2 / 5 m M A T P ( F i g . 2B). I n this c o n d i t i o n , the ( N a * + K * ) - A T P a s e appears to operate as an Na*-ATPase [73] and to exchange N a ' for N a * instead o f K * for N a ' [74,75], i n contrast to the N a * - N a * exchange process d r i v e n by A T P plus A D P where no net A T P hydrolysis seems to occur [76]. The ( A T P + A D P ) - s t i m u l a t e d N a * - N a * exchange is a well-described p a r t i a l reaction o f the ( N a * - I - K * ) - A T P a s e , occurring either i n p a r t i a l l y poisoned cells [77,78] or when N a * replaces the extracellular K * [ 7 9 - 8 4 ] . A p p a r e n t l y , the N a * ions are 'occluded' i n the ADP-sensitive E , f o r m o f ( N a * + K * ) - A T P a s e [82], w h i c h is able to accept the terminal phosphate f r o m A T P and to transfer it back to the A D P . The ( A T P + A D P ) - d r i v e n N a * - N a * exchange occurs also i n ( N a * -\ K * ) - A T P a s e liposomes w h i c h have exchanged their i n t e r n a i K ' p o o l for

N a * ions i n the p r é s e n c e o f external A T P , so that o p t i m a l c o n d i t i o n s for N a - N a exchange are p r é sent, i.e., high 'extracellular' (inside liposomes) N a * (100 m M ) a n d l o w 'extracellular' K * ( b e l o w I m M ) i n association w i t h the p r é s e n c e o f ' i n t r a c e l l u l a r ' (outside liposomes) A T P plus A D P [14,83]. T h e p r é s e n c e o f ' i n t r a c e l l u l a r ' K * does not hinder the N a * - N a * exchange process, b u t rather s t i m u l â t e s it [84], so that o p t i m a l c o n d i t i o n s are p r é s e n t i n the ( N a * + K * )-ATPase-hposomes w h e n the net N a * - K * exchange is finished [84,85]. T h e A T P - d e p e n d e n t ' u n c o u p l e d N a * transport' observed i n red cells i n d o w n h i l l c o n d i t i o n s [85] can be reproduced as u p h i l l N a * transport mediated by the K ' - f r e e ( N a * - ( - K * ) - A T P a s e i n l i p o somes ( F i g . 2B) reconstituted w i t h p u r i f i e d d o g k i d n e y ( N a * - l - K * ) - A T P a s e [86,87]. W h e t h e r this p a r t i a l reaction p r é s e n t s u n i d i r e c t i o n a l N a * transp o r t at a 0.5 N a * / 1 A T P r a t i o , w i t h empty sites m o v i n g i n the opposite d i r e c t i o n , o r a n exchange reaction where N a * substitutes for K * at the K * - b i n d i n g sites leading to a 3Na*-2Na*^ exchange can not be f i r m i y estabhshed o n the basis o f the tracer flux data. Beauge and Berberian [88] show that A T P - s t i m u l a t e d N a * uptake occurs also i n Na*-free liposomes c o n t a i n i n g choline, a l t h o u g h local recycling o f p u m p e d N a * can not be excluded w i t h certainty. Interestingly, acetyl phosphate can replace A T P for the K *-independent N a * uptake, i n d i c a t i n g that p h o s p h o r y l a t i o n at the catalytic site is sufficient to drive this p a r t i a l transport reaction [88]. A 3 N a - 2 N a exchange ratio seems n o t u n h k e l y i n inside-out red cell m e m b r a n e vesicles [75]. I f , i n the liposomes, three N a * ions were to move i n the u p h i l l d i r e c t i o n and t w o o f t h e m were i m m e d i a t e l y recycled back t h r o u g h the p u m p b y the t w o K * l o a d i n g sites, the overall s t o i c h i o m e t r y and k i n e t - ' ics w o u l d l o o k as i f one N a * i o n had m o v e d u n i d i r e c t i o n a l l y u p h i l l . Forgac and C h i n [86] also calculate f r o m the transmembranous [ • ' H ] t r i p h e n y l m e t h y l p h o s p h o n i u m i o n gradient that a positive inside p o t e n t i a l o f about 50 m V results f r o m the net N a * uptake, so u n d e r l i n i n g the electrogenic nature o f the N a * transport. By means o f an entrapped potential-sensilive fluorescent probe, Forgac a n d C h i n [87] further demonstrate that a p r o t o n efflux occurs i n the d i r e c t i o n opposed to the net N a * u p t a k e as w e l l as i n f l u x o f SO4, C l or

342

S C N anions. The p r o t o n efflux adjusts itself to the

periments w i t h reconstituted ( N a * - l - K * ) - A T P a s e

rate o f anion i n f l u x w h i c h , i n t u r n . d é p e n d s o n the

are reviewed elsewhere (e.g., Refs. 2 - 8 , 111, 112)

l i p i d - p e r m e a b i l i t y o f the selected anion [87]. A p -

so

parently, the net, electrogenic N a * flux associated

electrogenicity o f the p u r i f i e d ( N a * - ) - K * ) - A T P a s e

w i t h N a * ATPase activity carries along bidirec-

i n liposomes is discussed i n the p r é s e n t review.

t i o n a l p r o t o n and a n i o n movements,

that

o n l y the

récent

démonstrations of

the

presumably

I n small vesicles, the apparent N a : K flux r a t i o

to compensate i n part for the strong positive inside

is n o t an i n d i c a t o r for the electrogenicity o f the

potential b u i l t up b y the net N a * uptake.

p u m p . I n fact, i t can be estimated

that a

few

excess positive charges, i.e., less t h a n a 1% change IIIC.

Electrogenicity

associated

with Na ^-K *

ex-

change

i n the i n t e r n a i K * c o n c e n t r a t i o n , produce a transm e m b r a n o u s p o t e n t i a l d i f f é r e n c e of 85 m V [113]. Such a small change i n the i n t e r n a i c a t i o n c o n -

I n m a m m a l i a n u n m y e l i n a t e d nerve fibers [89],

c e n t r a t i o n is below the d é t e c t i o n l i m i t when iso-

cardiac Purkinje fibers [90], heart v e n t r i c u l a r [91]

topes are used to measure the change i n i n t e r n a i

or atrial [92] muscle, red b l o o d cells [93]. mouse

N a * and K * c o n c e n t r a t i o n , w h i c h means that i n

pancreatic B-cells [94], toad rods [95] and i n d i -

small vesicles i t is not possible to s e n s é the electro-

verse other tissues [96] it has been shown that the

genic c o m p o n e n t

sodium p u m p activity o f the ( N a * + K * ) - A T P a s e

contrast, o p t i c a l methods can detect 10 m V p o t e n -

System is accompanied

tials i n vesicles, corresponding t o a change i n the

by positive charge move-

ment, resulting f r o m N a * efflux

uncompensated

internai

f r o m the c a t i o n flux r a t i o . I n

K * concentration

o f o n l y 1-2

out

of

several h u n d r e d K * ions [113],

by K * i n f l u x [97.98]. T h a t more N a * ions than K * ions are

trans-

p o r t e d per p u m p cycle has been demonstrated i n red b l o o d cells, excitable

tissues, and

A s liposomes are t o o small for measuring transmembranous p o t e n t i a l

via

implanted

the élec-

epithelia

trodes, Chemical p o t e n t i a l i n d i c a t o r s , e.g., the l i p o -

[53,97]. The most frequently observed stoichiome-

p h i l i c a n i o n thiocyanate or fluorescent p o t e n t i a l -

try is close to 3 N a : 2 K : 1 A T P [ 4 - 6 , 8 , 5 3 ] .

sensitive

I n super- and perfused squid giant axons i t was shown that

the p u m p

electrogenicity associated

w i t h excess N a * flux appears to be

maintained

[100] also i n reversed c o n d i t i o n s where ions

are

substances,

are

used

to

monitor

the

p o t e n t i a l across the vesicle m e m b r a n e [114], F o l l o w i n g the a d d i t i o n o f external A T P to a ( N a * - I - K * ) - A T P a s e - l i p o s o m e suspension, there is a

concomitant

accumulation

of

the

lipophihc

r u n n i n g d o w n ] i i l l and A T P is synthesized b y the

lhio[''*C]cyanate a n i o n a n d the N a * c a t i o n , d e m -

( N a * + K * ) - A T P a s e System [101,102].

o n s t r a t i n g that the p u r i f i e d , reconstituted ( N a * - » -

The 3 N a * : 2 K * : 1 A T P s t o i c h i o m e t r y o f the

K ' )-ATPase retains its electrogenic p r o p e r t y and

( N a * + K.*)-ATPase is c o n f i r m e d b y t i t r a t i o n o f

créâtes

the

t r a n s m e m b r a n o u s t h i o [ ' ' ' C ] c y a n a t e gradient corre-

b i n d i n g sites located

on

the

(Na*-I- K * ) -

an

inside-positive

potential

[115].

ATPase m o l é c u l e w i t h labeled ligands. U s i n g v a r i -

sponds to a p o t e n t i a l o f 14 m V [115],

ons techniques to distinguish spécifie f r o m

p r é s e n c e o f n i g e r i c i n - an i o n o p h o r e for N a * and

non-

In

The the

indeed

K . * ions - the p o t e n t i a l decreases by 5 m V . T h i s

about three N a * - b i n d i n g sites and t w o K * - b i n d i n g

d é c l i n e is a t t r i b u t e d to the collapse o f the N a * and

specific l i g a n d - b i n d i n g , the authors f i n d

sites per p h o s p h o r y l a t i o n site [59,63,82,103-107]. F r o m kinetic studies i t seems that the N a * - d i s charge and

the

K *-loading sites coexist

rather

K*

diffusion

potentials.

In

conséquence,

the

p o t e n t i a l p r o d u c e d b y electrogenic i o n - p u m p i n g is about 9 m V . I n h i b i t i o n o f the active

transport

than being a single set o f sites alternating their

process by a p p l i c a t i o n o f vanadate to the c y t o -

affinity for N a * or K * ions [108].

plasmic p a r t o f the p u m p or o f o u a b a i n to the

I n view o f the well-established p u m p s t o i c h i o m etry i n cells, i t is not surprising that a 3 N a * : 2 K * transport r a t i o c o u l d be reproduced i n liposomes containing

purified,

functional

(Na*+K*)-

ATPase [16,17,109,110]. The early transport

ex-

extracellular p a r t abolishes the electrogenic potential [115]. I n the p r é s e n c e o f C l ~ ions, 1.6 to 2.2 N a * ions are taken

up per K * i o n extruded

[115]. C o n -

versely, i n the p r é s e n c e o f SO4 ions, the transport

343

ratio decreases to I N a * : I K * [115].

Presumably,

p l i n g r a t i o can be defined as follows [119]:

"the

C l " ions accompany the excess N a * ions to m a i n -

s t o i c h i o m e t r i c ratio, « , w h i c h is equal to the n u m -

tain electroneutrality d u r i n g the electrogenic w o r k -

ber o f i o n - b i n d i n g sites d i r e c t l y i n v o l v e d i n trans-

i n g o f the p u m p [115]. There is no i n d i c a t i o n i n

p o r t , should be distinguished f r o m the c o u p l i n g

the p u b l i c a t i o n o f D i x o n and H o k i n [115] as to

r a t i o , p. Whereas the c o u p l i n g r a t i o is variable and

whether the electrogenic activity o f the p u m p is

d é p e n d s o n the d r i v i n g forces, the s t o i c h i o m e t r i c

absent i n the neutral N a * - K * exchange mode.

r a t i o has a fixed value w h i c h is d e t e r m i n e d b y the

Nevertheless. the authors suggest that the 1 N a *-

transport mechanism. O n l y i n the Hmit o f perfect

I K * flux r a t i o may be o n l y apparent and that the

c o u p l i n g d o p and n become i d e n t i c a l " .

n o r m a l 3 N a : 2 K p u m p stoichiometry may be u n -

Presumably,

the

' perfect

c o n d i t i o n s ' are

en-

changed. Back-leak o f N a * ions w o u l d take care o f

countered b y the ( N a * - I - K * ) - A T P a s e i n a healthy

electroneutrality i n the sulfate liposomes, i.e., elec-

l i v i n g cell, i.e., ( i ) a high A T P / A D P : P, r a t i o , ( i i )

trogenicity c o u l d be p r é s e n t despite the apparently

l o w N a * a n d h i g h K * concentrations i n the i n -

neutral cation-exchange

tracellular c o m p a r t m e n t , ( i i i ) h i g h N a * a n d l o w

process. But h o w is i t

possible that N a * ions leak back r a p i d l y enough to

K ' concentrations i n the extracellular c o m p a r t -

reduce the 3 - 4 N a ' : 2 K * r a t i o to a I N a * : 1 K *

ment, as w e l l as ( i v ) intact p r o t e i n structure and

ratio? I t is indeed k n o w n that the passive N a *

(v) o p t i m a l l i p i d e n v i r o n m e n t . I n such c o n d i t i o n s

fluxes i n this System are at least 100-times slower

the c o u p l i n g r a t i o o f the ( N a * - I - K * ) - A T P a s e Sys-

than the active N a * fluxes and this is also the case

tem

in active transport c o n d i t i o n s [12,116]. T h e hy-

3Na*:2K*:l

pothesis o f a compensating N a * back-leak there-

c a t i o n o f one or o f several parameters c o n s t i t u t i n g

is

equal

to

the

stoichiometric

ratio

A T P [53]. T h e o r e t i c a l l y , m o d i f i -

fore implies that the passive N a * p e r m e a b i l i t y

the d r i v i n g force o f the transport process c o u l d

increases by a factor o f about 100 i n the sulfate

alter the N a * : K * : A T P c o u p l i n g r a t i o [119].

liposomes. But h o w is the p u m p then able to b u i l d up net cation gradients? O r does the

I n liposomes, the N a * : K * c o u p l i n g r a t i o i n -

membrane

creases f r o m 1.30 to 1.90 w h e n the N a C l a n d K.C1

potential i n the absence o f p e r m é a b l e C l " ions

concentrations i n the i n c u b a t i o n m é d i u m are 30

force

m M and 50 m M , respectively [16,115]. W h e n the

the excess N a *

ions

to flow

backwards

through the p u m p m o l é c u l e itself?

N a C l c o n c e n t r a t i o n is raised f r o m 25 to 75 m M

T o m o n i t o r c o n t i n u o u s l y the electrogenic p u m p

and

the

K C I concentration

is

simultaneousiy

component, recording the fluorescence signal o f a

lowered f r o m 75 to 25 m M , the c o u p l i n g r a t i o

voltage-sensitive

choice

increases f r o m 0.8 N a * to 1.8 N a * per K * , sug-

[113,114]. The indodicarbocyanine dye has t u r n e d

gesting that the N a * : K * c o n c e n t r a t i o n r a t i o i n

dye

is

the

method

of

out to be a sensitive i n d i c a t o r o f the electrogenic

the m é d i u m influences the c o u p l i n g r a t i o to some

component associated w i t h the transport a c t i v i t y

extent [83]. I n agreement

of the reconstituted ( N a * - l - K * ) - A T P a s e [117,118].

Blostein [120] demonstrates w i t h inside-out vesicles

w i t h this observation,

be

made f r o m red b l o o d cells that the N a * : K * c o u -

directly and continuously registered i n a f l u o r i m -

p l i n g r a t i o is between 0.44 and 0.53 at 0,18 m M

The transport rate o f the s o d i u m p u m p can

eter [117,118]. U s i n g the f l u o r i m e t r i c technique, an

NaCl

activation energy o f 115 k j / m o l ( = 27 k c a l / m o l )

comes

and 0.2 m M R b C l , respectively, and 1,22

N a * : I K * when

the

external

be( =

for the active transport process was f o u n d . T h e

cytosolic) N a * c o n c e n t r a t i o n is increased

e q u i l i b r i u m dissociation constants for the depen-

to 1,80 m M w h i l e the R b C l c o n c e n t r a t i o n remains

10-fold

dence o f the active transport rate o n the A T P

at 0.2 m M [134]. Conversely, G o l d i n [17] observed

high-affinity

stable 3 N a * : 2 K * c o u p h n g ratios w h e n the K *

A T P site and 0.15 m M for the l o w - a f f i n i t y A T P

c o n c e n t r a t i o n was raised f r o m 11 to 30 m M at 30

site [117,118].

mM

concentration were 0.7 ^iM

for the

N a * , O b v i o u s i y , the p r é c i s e effect

N a * : K * concentration IIID.

Chemical modification

The

relationship

of active

between

the

transport stoichiometric

ratio o f an active transport process and the c o u -

ratio on

the

of

the

Na* : K *

c o u p l i n g r a t i o i n the reconstituted S y s t e m remains to be clarified. Experiments s t u d y i n g a possible r é g u l a t i o n o f

344

ihe N a " : K * c o u p l i n g ratio by N a * and K * c o n -

1.5 to 2 N a * per K * are transported

centrations y i e l d also c o n t r a d i c t o r y results i n cells.

[132]. ( F o r an excellent review o f N a * : K * cou-

I n E h r l i c h ascites t u m o r cells [121] and i n squid

p l i n g ratios see Réf. 98,) O n the other hand, such d é v i a t i o n s f r o m

axons [122] for instance, the N a * : K * c o u p h n g ratio varies as a f u n c t i o n of the N a * a n d

K*

i n muscle

the

3 N a * : 2 K * stoichiometry c o u l d s i m p l y resuit f r o m

concentrations s u r r o u n d i n g the membrane. I n con-

the statistical v a r i a t i o n o f the t r a n s p o r t measure-

trast, no such effect was seen i n red b l o o d cells

ments. I t is d i f f i c u l t

[123].

1.5Na* : I K * and 2 N a * : I K * ratios i n a statisti-

to distinguish

lNa*:IK*,

I n liposomes, N a ' : K * c o u p l i n g ratios v a r y i n g

cally significant way. Indeed, the e x p é r i m e n t a l de-

between 1 and 2 N a * per K * are observed also at

sign is not easy, as b o t h N a * and K * fluxes should

fixed N a C l and

be measured simultaneously i n the i n i t i a l transport

K C l concentrations.

N a C l and the K C l concentrations

When

the

are, for exam-

phase where

the flux rates are Hnear and

then

ple, 30 m M each, the N a * : K * c o u p l i n g ratios

expressed i n absolute a m o u n t s o f i o n transported

range between

and

w h i c h requires p r é c i s e knowledge o f the N a * a n d

Pick [110] show a 3 N a * : 2 K * ratio i n the experi-

K * concentrations o n b o t h sides o f the membrane.

1.61 and 2.22 [115]. K a r h s h

ment shown i n their F i g . 4 and a I N a * : I K * r a t i o in the experiment o f their F i g . 10. S i m i l a r l y , w i t h

T h a t , besides the N a * and K * the

structure

and

conformation

concentrations, o f the ( N a * +

N a * and K * concentrations fixed at 50 m M each,

K * ) - A T P a s e p r o t e i n influences the N a * : K * c o u -

the N a * : K * c o u p l i n g ratios vary f r o m

roughly

p h n g ratio can be d e m o n s t r a t e d d i r e c t l y i n the

Moos-

liposomes b y r e c o n s t i t u t i n g i n p a r a l l e l c o n t r o l and

lNa*:lK*

to 2 N a * : l K * ( A n n e r

mayer, unpublished

results).

and

Presumably,

varia-

tions i n the p r o t e i n / l i p i d ratio, l i p i d c o m p o s i t i o n

selectively m o d i f i e d enzyme a n d

comparing

the

transport pattern o f b o t h p r é p a r a t i o n s .

and i n the structural i n t e g r i t y of the enzyme affect

One o f the best-described m o d i f i c a t i o n s of the

the N a * : K * c o u p l i n g ratio, at least i n the Hpo-

( N a * + K * ) - A T P a s e p r o t e i n is s é l e c t i v e p r o t e o l y -

somes. Whether nature uses such parameters to

sis o f the N a * f o r m

modulate the N a * : K * c o u p h n g ratio is not yet

enzyme. Somogy [133] observed a protective effect

k n o w n , but the fact that c o u p l i n g ratios r a n g i n g

o f N a * , K * or M g ^ * w i t h regard to proteolysis by

f r o m 0.5 to 3 N a * per K * ( F i g . 3) are f o u n d b y

t r y p s i n o f ( N a * - I - K * ) - A T P a s e a n d proposed that

différent

the p r o t e c t i o n reflects c a t i o n - i n d u c e d

authors [98] may be due

to such cir-

cumstantial variations. F o r instance, i n red b l o o d cells, N a * : K * transport

ratios ranging

or o f the K f o r m o f

the

conforma-

t i o n a l changes of the ( N a ' -I- K * ) - A T P a s e p r o t e i n .

between

Jorgensen [134] analyzed

the p u t a t i v e

confor-

1 : 1 [ 1 2 4 - 1 2 6 ] , 1 . 2 - 1 . 3 5 : 1 [127], 1.5:1 [123] and

mational

2 : 1 [128] were f o u n d . I n nerve fibers, ratios o f

showed that, i n the p r é s e n c e o f N a * , the ( N a * - I -

change

with

careful

experiments

and

1.33 to 1.5 : I K * [100], 2 - 3 N a * : l K * [129], or

K * )-ATPase

variable ratios [130,131] were reported. S i m i l a r l y ,

kinetics: - a 20 m i n r a p i d linear phase f o l l o w e d b y

a c t i v i t y decreases w i t h

two-phase

a slow Hnear phase - whereas i n the p r é s e n c e o f K*

the enzyme a c t i v i t y disappears w i t h

single-

phase kinetics. Such distinct i n a c t i v a t i o n kinetics i m p l y a c a t i o n - i n d u c e d change i n the exposure o f c r u c i a l trypsin-sensitive b o n d s o f the ( N a * - I - K * ) A T P a s e a-subunit.

K

W h e n the digestion o f the N a ^ f o r m is stopped b y the a d d i t i o n o f soybean t r y p s i n i n h i b i t o r at the end o f the r a p i d phase, a stable, m o d i f i e d ( N a * + K * ) - A T P a s e f o r m can be isolated w i t h an intact 1-6

Fig. 3. Apparent coupling ratios of the (Na* + K * )-ATPasemediated N a * - K * exchange process in cells, vesicles and liposomes. Références in the lext (subsection IIID).

number about

of phosphorylation 50%

o f the

sites b u t w i t h o n l y

original ( N a * - H K*)-ATPase

a c t i v i t y [135,136]. T h e p h o s p h o f o r m

o f this ' i n -

v a l i d ' enzyme is A D P - s e n s i t i v e instead o f being

345

K *-sensitive, a ' s y m p t o m ' i n d i c a t i n g that the p u m p cycle is interrupted at the E i - E j t r a n s i t i o n step [135]. W h e n such selectively m o d i f i e d , i.e. ' i n v a l i d ' ( N a ' + K * )-ATPase is reconstituted i n liposomes, its N a * transport capacity is decreased b y about 50% as compared to the N a * transport o f the reconstituted c o n t r o l enzyme, whereas the overall K *-extrusion process appears to be unchanged [137,138]. W h e n the enzyme is further digested, the N a *-transport capacity continues to decrease i n parallel w i t h the ( N a * + K * ) - A T P a s e a c t i v i t y [139]. However, i n contrast to the i n i t i a l 20 m i n phase, where the K transport appears unchanged, the entrapped K * pool decreases i n the second inactivation phase and this also i n parallel w i t h the d i m i n u t i o n of the enzyme a c t i v i t y and the N a * transport [139]. N o proteolytic split is seen i n the ' i n v a l i d ' enzyme, whereas i n the second digestion phase o f the N a * form, fragments resulting f r o m spécifie proteolytic splits can be seen after s é p a r a t i o n and staining of the a and fi subunits o n polyacrylamide gels i n SDS [134.135,139]. The fact that the degree of proteolysis of the a-subunit is q u a n t i t a tively related to a decrease i n K ^ entrapment b y the liposomes reconstituted w i t h the degraded enzyme indicates that the proteolytic 'splits' o f the isolated ( N a * + K * )-ATPase become ' leaks' i n the liposomes [139.140] (see Section I V for discussion of the ( N a * - ) - K * ) - A T P a s e leakage channel i n (Na*-I- K*)-ATPase). When trypsin is added directly to liposomes reconstituted w i t h intact ( N a * + K * ) - A T P a s e , b o t h N a * and K * transport decrease simultaneously and the t y p i c a l transport pattern o f the ' i n v a l i d ' enzyme, i.e., r é d u c t i o n of about 50% ATPase activity logether w i t h 50% N a ' transport, resulting i n a decreased N a * : K * c o u p l i n g ratio, is not seen [110], p r o b a b l y because of the starting l o w l N a * : l K * c o u p l i n g ratio, w h i c h c o u l d be due, as previousiy suggested [139], to the p r é s e n c e of the soybean trypsin i n h i b i t o r - t r y p s i n m i x t u r e . A m i x t u r e of the i n h i b i t o r w i t h t r y p s i n r e t a i n i n g some residual proteolytic a c t i v i t y could produce a split so close to the end o f the a-subunit a m i n o acid s é q u e n c e that i t can n o t be seen b y gel electrophoresis and could so f o r m ' i n v a l i d ' enzyme w i t h a lowered N a * : K * c o u p l i n g ratio as c o m pared to untreated c o n t r o l enzyme. I f this inter-

p r é t a t i o n were correct, the results obtained w i t h pretreated ( N a * - K K * ) - A T P a s e [ 1 3 7 - 1 4 0 ] o r b y a d d i n g t r y p s i n externally to intact ( N a * + K * ) A T P a s e liposomes [110] w o u l d a g r é e perfectiy and c o u l d b o t h be interpreted by a first effect o f a putative very l i m i t e d proteolysis (no fragments visible o n gels) p r o d u c i n g ' i n v a l i d ' enzyme w i t h a I N a * ; I K * r a t i o [110,137-140] f o l l o w e d by a seco n d phase w i t h visible p r o t e o l y t i c splits where N a * and K * transport c o n c o m i t a n t l y decrease. I f l i m i t e d t r y p s i n treatment had a spécifie effect o n the E1-E2 t r a n s i t i o n rate, leading to freezing o f the K*-insensitive E, f o r m , one w o u l d predict a s él ect i v e r é d u c t i o n o f the K * transport capacity. Y e t , another spécifie enzyme m o d i f i c a t i o n leading to a decreased E ^ - E j t r a n s i t i o n rate, i.e., sél ect i v e b l o c k i n g o f S H groups by A'-ethylmaleimide (e.g. Refs. 141, 142), displayed the same sélective r é d u c t i o n o f the N a * - t r a n s p o r t capacity after recons t i t u t i o n i n t o liposomes [140]. substantiating the concept that a reduced rate o f this critical conform a t i o n a l change is associated p r i m a r i l y w i t h a d i m i n i s h e d reduced N a *-transport capacity o f the enzyme. T h e p r é s e n c e o f a labile N a * - l r a n s p o r t f r a c t i o n is documented also b y Pennington and H o k i n [116] w h o demonslrate that wheat-germ a g g l u l i n i n b o u n d l o the glycosylated parts o f ( N a * - l - K * ) A T P a s e reduces the c o u p l i n g r a t i o from 3 N a * : 2 K * to I N a * : 1 K * . A t first sight, i t w o u l d be more logical i f the decreased K *-sensitivity o f the frozen E , conform a t i o n were expressed as lowered K * transport. However, an arrest somewhere i n the p u m p cycle can affect almost any cation l o a d i n g or dischargi n g step (e.g.. Réf. 143). Thus, m o d u l a t i o n o f the E , - E 2 t r a n s i t i o n rate either by chemical m o d i f i c a t i o n or b y changing the degree o f the c a t i o n site o c c u p a t i o n b y altered c a t i o n concentrations c o u l d m o d u l a t e the System i n a way i n w h i c h i t exchanges o n l y one N a * i o n per K * i o n instead o f t w o , three or even f o u r N a * ions. Such p a r t i a l l o a d i n g o f ihe Na*-sites ( F i g . 3) c o u l d explain the 0 . 5 N a * : 1 K * r a t i o observed i n inside-out vesicles at a l o w N a * : K * c o n c e n t r a t i o n r a t i o [120], the l N a * : l K * r a t i o occasionally observed i n cells [ 1 2 4 - 1 2 6 ] o r liposomes [83,115], the 'classical' 3 N a * : 2 K * r a t i o i n cells [53] or liposomes, [16,17,109] as w e l l as the 2 N a * : I K * r a t i o [83,115,129].

346

Whether protons replace the N a * i o n at

the

e m p t y N a * sites and perhaps also at p o t e n t i a l l y

externally after r e c o n s t i t u t i o n , f o r instance, vanadate [137,138]. T h a t vanadate is an i n h i b i t o r acting d i r e c t l y at

empty K * sites, as suggested f r o m an effect o f protons o n the affinity o f the enzyme for N a * and

the intracellular p a r t o f the pure s o d i u m

K * ions [71,144]; and f r o m a p r o t o n efflux o b served d u r i n g ' u n c o u p l e d N a ' transport' [87] is

liposomes as a t o o l [137,138,151]. N o t o n l y does

not yet k n o w n .

vanadate b l o c k the active transport process. but

Such

detailed

déterminations

of

'abnormal'

demonstrated

using

pump

was

(Na*-I-K*)-ATPase-

the decrease o f the vanadate sensitivity by l i m i t e d

N a * : K ' c o u p h n g ratios w i l l have i m p o r t a n t i m -

proteolysis [152,153] can

plications w i t h regard to the transport mechanism

reconstituted System and is expressed as relatively

o f the p u m p and may help to answer the f o l l o w i n g

vanadate-resistant

type o f question:

vanadate concentrations

A r e we dealing with 'sequential r o t a t i n g site' m o d -

reconstituted ( N a * + K * ) - A T P a s e are close to 1

els, where [ 1 4 5 - 1 4 7 ] the

mM

transport

molécule

is

be reproduced

Na*

transport

in

[137],

the The

necessary to block

the

[151], because the i n h i b i t o r was added

to

equipped w i t h three c a t i o n - b i n d i n g sites that are

actively p u m p i n g liposomes i n c u b a t e d i n the p r é s -

exposed to the cytoplasmic m é d i u m to b i n d three

ence o f at least 20 m M N a C l , and N a * ions are

N a * ions d u r i n g p h o s p h o r y l a t i o n o f the

i n h i b i t o r y f o r vanadate b i n d i n g [154,155] w i t h an

enzyme

p r o t e i n by A T P whereupon the sites rotate across

/JO

the membrane

vanadate concentrations are required to d e m o n -

to exchange the three N a *

ions

around

7

mM

[156].

That

relatively h i g h

strate transport i n h i b i t i o n i n active p u m p c o n d i -

against two K * ions? O r are we dealing w i t h

t w o sets o f coexisting

tions, i.e., i n the p r é s e n c e o f N a * ions, has been

c a t i o n - b i n d i n g sites: a set o f Na*^-binding sites at

observed

the inner side o f the membrane

a set o f

l i p o s o m e p r é p a r a t i o n s [110] or i n red cells [157],

and

also

in

other

( N a * + K*)-ATPase-

membrane

squid axons [158] a n d epithelia [159]. I n contrast,

surface? I n this situation, the N a * and K * ions

v a n a d a t e - b i n d i n g studies o n p u r i f i e d enzyme can

have the o p p o r t u n i t y to be transported simulta-

be p e r f o r m e d i n the absence o f N a * ions w i t h

neously. Sequential and simultaneous p u m p m o d -

vanadate concentrations i n the n a n o m o l a r

els are compared i n the review o f G a r r a h a n

[138,104].

K * - b i n d i n g sites exposed at the outer

and

range

Cornehus and Skou [160] make use o f the asym-

Garay [148]. O r w i t h transmembrane channel structures [66]

m e t r i c a l o u a b a i n and vanadate i n h i b i t i o n to d é -

equipped w i t h appropriate energy barriers [149]?

termine the o r i e n t a t i o n o f the reconstituted ( N a *

I n fact, m o d e m molecular studies o f the properties

+ K*)-ATPase

of

l i p i d / p r o t e i n r a t i o a n d o n the l i p i d c o m p o s i t i o n ,

the transmembrane

( N a * - l - K * ) - A T P a s e and

molécules.

Depending

other membrane transport proteins establish that

they f i n d enzyme d i s t r i b u t i o n s r a n g i n g

transmembrane

10-26%

proteins d o not f l i p flop (rotate)

across the membrane

-

at least not w i t h

constants c o m p a t i b l e

with

transport

time

processes.

inside-out,

10-43%

on

the

between

non-oriented

and

4 9 - 6 5 % right-side-out. K a r l i s h et a l . [51,52] use differential

vanadate i n h i b i t i o n

to describe

the

Subtle c o n f o r m a t i o n a l changes i n the 0.2-0.3 n m

sidedness and mechanism o f the K * : K * exchange

range are sufficient to account for active, p r o t e i n -

mechanism. A n o t h e r ( N a * - I - K * ) - A T P a s e i n h i b i -

mediated i o n transport and the transport m o l é c u l e

t o r w h i c h blocks the N a * : K * exchange process

possibly o s c i l l â t e s between t w o e x t r ê m e c o n f o r m a -

f r o m the c y t o p l a s m i c side, i.e., at the external side

tional

of

States

reguialed

by

ATP-induced

phos-

p h o r y l a t i o n and d e p h o s p h o r y l a t i o n [150], occlud-

the

reconstituted

( N a * - I - K * )-ATPase-

liposomes is the A T P - a n a l o g u e , C r - A T P [161]. F i n a l l y , the reconstituted s o d i u m p u m p can be

i n g alternately N a * or K * ions. The effect and the sidedness o f well-described

' m o d i f i e d ' by d i l u t i n g the p u m p m o l é c u l e s i n the

(Na*-I- K * ) - A T P a s e i n h i b i t o r s o n the active trans-

l i p i d phase to decrease p r o t e i n - p r o t e i n i n t e r a c t i o n .

port

I f p r o t e i n aggregation were to p l a y a r ô l e i n m o d -

process,

e.g.,

of

cardioactive

steroids

[16,17,109,111], can be studied b y i n c l u d i n g the

u l a t i n g the transport

chemicals w i t h i n the hposomes or a d d i n g

large

them

number

of

process, liposomes w i t h

potentially

interacting

a

pump

347

m o l é c u l e s should display a transport p a t t e r n dif-

o f leaky vesicles is f o r m e d w h e n the altered en-

férent to that obtained f r o m liposomes c o n t a i n i n g

z y m e is reconstituted i n t o liposomes. A s i n the

only

case

one

pump

molécule.

The

fact

that

the

of

reconstituted

proteolyzed

( N a * - F K . ' )-

N a *^ : K * exchange rate increases linearly w i t h the

ATPase, the p r o p o r t i o n o f d i s r u p t e d vesicles corre-

number o f p u m p m o l é c u l e s per liposomes [42,162]

sponds to the fraction o f inactivated ( N a * - I - K * ) -

precludes intermolecular but not i n t e r s u b u n i t [163]

ATPase.

interaction.

T h e fact that a single chemical m o d i f i c a t i o n i n the ( N a * + K * ) - A T P a s e m o l é c u l e decreases

I V . Leakage channel

enzyme a c t i v i t y and

the

at the same t i m e forms a

leakage channel indicates that a m i n o r structural IVA.

In

liposomes

a l t é r a t i o n unmasks a leakage p a t h w a y b y uncoup l i n g not o n l y the catalytic energy transformer but

I n g ê n e r a i , reconstituted membrane vesicles are composed

by tight and

leaky p o p u l a t i o n s .

For

instance, only 1 out o f 4 - 6 vesicles is sealed i n a

also the g a t i n g c o m p o n e n t

from

a

nonselective

channel. L i n g and N e g e n d a n k

[168] speculate that

the

p r é p a r a t i o n from m a m m a l i a n brush-border vesicles

d i f f é r e n t ^ " N a * , '^'^Rb* o r ^ ^ K * contents observed

[164] and 1 out o f 2 sarcoplasmic r e t i c u l u m vesicles

i n the vesicles after a d d i t i o n o f A T P are due to

[165].

A T P - m o d u l a t e d leaks o c c u r r i n g d u r i n g the wash

Liposomes reconstituted w i t h b a n d 3 p r o t e i n

process i n the gel c o l u m n s . T h e y suggest

that,

and egg PC c o n t a i n 6 n m pores w h i c h are absent

w h e n the liposomes had been incubated i n the

when the liposomes are f o r m e d w i t h the o r i g i n a l

p r é s e n c e o f A T P before

erythrocyte Hpid, suggesting that t h è s e p a r t i c u l a r

w o u l d leak out i n t o the c o l u m n whereas N a * ions

pores may be due to defective Hpid p a c k i n g at the

w o u l d r e m a i n enclosed i n the liposomes d u r i n g gel

p r o t e i n / l i p i d interface [166].

f i l t r a t i o n . T h e opposite w o u l d be v a l i d for l i p o -

gel f i l t r a t i o n .

K*-ions

Defective Hpid p a c k i n g is not the p r i n c i p a l cause

somes that had not been i n contact w i t h A T P . I f

o f pore f o r m a t i o n i n ( N a * - l - K * ) - A T P a s e l i p o -

this hypothesis were true. the p r é s e n c e o f A T P i n

somes. Several lines o f é v i d e n c e locale the leakage

the c o l u m n should prevent

channel

and

^~Na* f r o m liposomes that had been i n c u b a t e d

that

w i t h ^ ^ N a * i n the absence o f A T P . H o w e v e r . n o

i n the

Caldenty

p r o t e i n structure.

[167] demonstrate.

for

Wheeler instance,

lipid-depleted ( N a * - l - K * ) - A T P a s e induces

path-

the ioss o f i n t e r n a i

such effect is observed w h e n ~ - N a " - l o a d e d

lipo-

ways for ['''C]sucrose i n PS liposomes. T h e pore-

somes are washed by passage t h r o u g h Sephadex-

f o r m a t i o n can be reproduced

G - 5 0 i n the p r é s e n c e or absence o f A T P ( A n n e r

indicating

that

the

by T r i t o n

X-100,

lipid-depleted ( N a * + K * )-

and

Moosmayer,

unpublished

data).

Likewise.

H o k i n et al. [15] observe no effect o f A T P o n

ATPase has a detergent-like a c t i o n [167]. Vesicle disrupture occurs also w h e n

trypsin-

^^Na*-loaded Hposomes d u r i n g their e l u t i o n i n a

treated ( N a * - l - K * ) - A T P a s e is reconstituted i n t o

Sephadex-column.

80% egg-PC-20% PS liposome [139,140]. T h e frac-

" ^ R b * t r a p p i n g is not affected by the

tion o f disrupted vesicles is directly related to the

used for l i p o s o m e wash b y gel f i l t r a t i o n . F o r i n -

fraction o f enzyme that has been inactivated b y

stance, passage t h r o u g h Sephadex G-50 c o l u m n s

trypsin treatment

for 4 - 6

[139,140], suggesting

that

the

F u r t h e r , the - ~ N a * . ''^K.*^

or

techniques

m i n i n the p r é s e n c e o f N a " . K * and

critical proteolytic split is expressed as a leakage

M g ^ * ions at r o o m t e m p é r a t u r e [15] o r i n buffer

channel after r e c o n s t i t u t i o n i n t o liposomes.

w i t h o u t M g - * , N a * a n d K * ions at 2 ° C [169] o r drastic

r a p i d f i l t r a t i o n ( i n a centrifuge) t h r o u g h syringes

structural a l t é r a t i o n o f the ( N a * - I - K * ) - A T P a s e

f i l l e d w i t h Sephadex gel [170], o r passage t h r o u g h

T h e leak f o r m a t i o n does not require

m o l é c u l e . W h e n the enzyme is incubated for 30

ion-exchange c o l u m n s i n sucrose for 30 s [110,171]

m i n at r o o m t e m p é r a t u r e i n the p r é s e n c e o f m i l l i -

y i e l d essentially the same entrapped isotope c o n -

m o l a r concentrations o f vanadate [42] or C r - A T P ,

centrations despite the drastically d i f f é r e n t e l u t i o n

an A T P analogue ( A n n e r . B . M . , M o o s m a y e r . M .

methods. Therefore. it w o u l d be surprising i f the

and Schoner, W., unpublished data), a p o p u l a t i o n

gel f i l t r a t i o n p r o c é d u r e were the c r i t i c a l step w i t h

348

regard to isotope t r a p p i n g i n tight, actively transp o r t i n g ( N a " + K ' )-ATPase hposomes. Moreover, variations of the e x p é r i m e n t a l conditions used for the transport assay before the washing step p r o duce flux kinetics w h i c h are entirely consistent w i t h theoretical p r é d i c t i o n s [54], so that L i n g ' s idea [168] of r a t i o n a l i z i n g a i l observations made i n reconstituted ( N a * + K A T P a s e liposomes b y differential artifactual leaks occurring d u r i n g the removal o f external isotope can now definitely be discarded. I n a d d i t i o n to this unequivocal e x p é r i m e n t a l d é m o n s t r a t i o n , there are t w o theoretical arguments i n v a l i d a t i n g Ling's hypothesis w i t h regard to the reconstituted ( N a * - l - K * ) - A T P a s e . F i r s t l y . if the presumptive A T P - i n d u c e d t r a n s i t i o n f r o m a folded, N a * - b i n d i n g , to an extended, K * - b i n d i n g protein structure were to be the r a t e - l i m i t i n g step [172]. the kinetics o f this process should be i n the range of seconds to minutes to account f o r the active transport kinetics. However. the possible physicai nature o f such a slow c o n f o r m a t i o n a l change has not yet been described [168,172]. Secondly. it has been shown experimentally that there are only 2 - 3 K ions b o u n d per ( N a * + K * ) ATPase m o l é c u l e after gel f i l t r a t i o n [59,82] o r after washing of the enzyme b y centrifugation p r o c é d u r e s [107]. I n contrast, liposomes c o n t a i n i n g only one ( N a * + K * ) - A T P a s e m o l é c u l e retain several thousand K * ions when the same washing conditions are applied, w h i c h i l l u s t r â t e s that the K * ions extruded by the A T P - a c t i v a t i o n o f the inside-out-oriented p u m p m o l é c u l e s are contained w i t h i n the aqueous vesicle space and are not b o u n d to the ( N a * + K * ) - A T P a s e p r o t e i n as postulated by Ling's association-induction hypothesis. I n deed. i f the latter were the case, the a m o u n t of N a ' or K * ions retained i n ( N a * + K * ) - A T P a s e liposomes should d é p e n d on the q u a n t i t y of p r o tein incorporated per vesicle. However, it has been shown experimentally that the number of ions retained i n the vesicles is the same whether the vesicle contains t w o , three or five p u m p m o l é c u l e s [42]. Thus, i n c o n t r a d i c t i o n w i t h L i n g ' s hypothesis, the amount o f trapped N a * or K * ions d é p e n d s on the vesicle volume and not o n the q u a n t i t y o f protein incorporated per vesicle. Taken together, the kinetic and q u a n t i t a t i v e results o b t a i n e d with (Na* + K*)-ATPase-

liposomes can be explained by a r a t e - l i m i t i n g step at the membrane b u t are d i f f i c u l t to reconcile w i t h L i n g and Negendank's s p é c u l a t i o n [168,172] o f s él ect i v e i o n leaks d u r i n g liposome wash. A f t e r a i l , L i n g ' s proposai that A T P is a ' c a r d i n a l adsorbant' i n d u c i n g a c o n f o r m a t i o n a l change i n the p r o t e i n leading to sél ect i v e i o n b i n d i n g corresponds i n m a n y aspects to the molecular mechanism p r o posed for the s o d i u m p u m p [ 4 - 6 , 4 4 ] . Conseq u e n t l y , at least w i t h regard to the ( N a * - I - K * ) ATPase-molecule, the apparent discordance between Ling's association-induction theory and the p u m p theory [173] is i n part a p r o b l e m o f terminology. / VB. In planar

bilayers

Several laboratories report a conductance i n crease i n black l i p i d membranes by the s i m u l t a neous p r é s e n c e o f ( N a * -I- K * )-ATPase, A T P , N a * , K * and M g ' * at the cis side o f the bilayer; the trans side becomes then electrically positive [ 1 7 4 - 1 7 7 ] . T h i s is taken as é v i d e n c e for the electrogenic a c t i v i t y resulting f r o m an N a * : K * transp o r t ratio exceeding u n i t y [ 9 7 - 9 9 ] . However, a i l authors a g r é e that more w o r k is required to i n t e r pret the bilayer results d e f i n i t e l y as é v i d e n c e o f p u m p electrogenicity. H y m a n [178], for instance, sees the 'electrogenicity' i n black l i p i d membranes b y a d d i t i o n o f A T P and acidic Hpids, e.g., cardioHpin. He infers that the short-circuit current results from a "surface p h e n o m e n o n p r o b a b l y due to alignment of A T P o n the p h o s p h o l i p i d b y i o n association at its interface w i t h the water phase". Shamoo and A l b e r s [179] demonstrate that an acid-soluble fraction o f a t r y p t i c digest from ( N a * + K * ) - A T P a s e p r é p a r a t i o n s increases black l i p i d membrane conductance. Shamoo and c o l l a b o r a tors [180] later i d e n t i f y a neutral fragment o f the small p o l y p e p t i d e subunit w i t h s o d i u m i o n o p h o r e a c t i v i t y . Tosteson and Sapirstein [181] d e m o n strate that p r o t e o l i p i d s o f the same type as f o u n d i n p u r i f i e d ( N a * - l - K * ) - A T P a s e p r é p a r a t i o n s [182] display single-channel behaviour o f 10 to 100 pS conductance i n K C l . Whether the p r o t e o l i p i d s are identical w i t h c e r t a i n t r y p s i n fragments remains to be estabhshed by a m i n o acid sequencing. F o r single-channel recording, a single ( N a * - I K * ) - A T P a s e m o l é c u l e can be i n t r o d u c e d i n t o b i -

349

layers by fusion o f well-characterized ( N a * + K ' )ATPase liposomes c o n t a i n i n g o n the average one p u m p m o l é c u l e per vesicle [183]. Single-channel recording reveals conductance o f about 40 pS. W h e n enzyme w i t h a digested a-subunit is i n t r o duced in the bilayer, the leakage channel is still p r é s e n t [183], indicating that it is constituted presumably by the trypsin-resistant h y d r o p h o b i c fragments or by the ^ - s u b u n i t . Incorporation o f a p u r i f i e d ( N a ' + K ' )-ATPase p r é p a r a t i o n i n t o the planar bilayer yields channels appearing i n clusters w i t h an ouabain- a n d vanadate-sensitive conductance o f u p to 250 pS [184]. Presumably, the decane i n the bilayer exposes the nonspecific channel component o f the ( N a * + K ' )-ATPase m o l é c u l e . Whether decane acts i n d i rectiy via increase o f bilayer thickness [185] o r directly w i t h the p r o t e i n part o f the p u m p m o l é cule is not k n o w n . I l also remains t o be explained h o w vanadate and ouabain close the leakage channel o f the ( N a * + K * )-ATPase m o l é c u l e s i n the planar bilayer i n a c o o p é r a t i v e fashion [184]. M i r o n o v et a l . [186] observe that the large subunit alone forms a channel that is regulated by A T P i n the p r é s e n c e o f M g ^ * , whereas Sorokina [187] .sees n o A T P effect. A i l authors. however, a g r é e that the c o n d u c t i v i t y is blocked by ouabain and vanadate as long as the m o l é c u l e is intact. T h e channels formed b y bromocyan fragments o f the ( N a ' + K * ) - A T P a s e protein n o longer respond to the p u m p i n h i b i t o r s [188], inferring that the functional components o f the p u m p m o l é c u l e must be able to interact a m o n g them.selves to close the conductance channel, perhaps by i m m o b i l i z i n g the gating c o m p o n e n t o r b y sealing it w i t h the catalytic part o f the (Na*-»K * )-ATPase m o l é c u l e . JVC.

Models

Shamoo a n d G o l d s t e i n [20] propose a m o d e l i n which the N a * - i o n o p h o r e o f the small p o l y p e p t i d e carries N a * ions i n t o a hollow, helical transmembraneous segment o f the large p o l y p e p t i d e w h i c h transfers the N a * ions t o the opposite side of the membrane b y an ion-exchange mechanism. The authors suggest that ATPase pumps c o n t a i n three components: a large non-selective channel, an ion-selective g â t e and an energy transducer. Likewise, Kagawa [189], g i v i n g H ' - A T P a s e as an

example, proposes that active transport Systems are composed by channel, gating u n i t , a n d energy transformer subunits. Such a gated channel c o m ponent i n the ( N a * H- K * )-ATPase m o l é c u l e c o u l d explain the voltage-induced, ouabain-sensitive channels seen i n h u m a n erythrocytes by T s o n g and collaborators [190]. It is reasonable to assume that the three funct i o n a l components o f the ( N a * + K ' )-ATPase m o l é c u l e , i.e., channel. gating u n i t a n d catalytic parts can be uncoupled a n d recoupied under appropriate e x p é r i m e n t a l conditions. I n the absence o f A T P , the slow passive K * a n d N a * fluxes mediated b y the dephosphoenzyme i n liposomes [ 1 1 - 1 4 ] c o u l d express activity o f the gated channel u n i t u n c o u p l e d f r o m the catalytic part. A c c o r d i n g l y , the leakage-channel o f proteolyzed reconstituted enzyme m a y be the functional expression o f the ungated. i.e., non-selective channel component. W i t h regard t o the three c o m p o n e n t p u m p model proposed by Shamoo a n d G o l d s t e i n [20] and Kagawa [189], it can be speculated that some N a * o r K * channels i n the cell membrane c o u l d be i n c o m p l è t e forms o f p u m p m o l é c u l e s that c o n t a i n the leakage channel w i t h o r w i t h o u t the gating c o m p o n e n t b u t w i t h o u t the energy transducer req u i r e d to p e r f o r m u p h i l l i o n transport. Perhaps. i n é v o l u t i o n , the p r i m i t i v e cell started w i t h simple, ion-specific ionophores that were then perfected t o ionophores capable o f exchanging similar ions passively a n d were f i n a l l y c o u p l e d t o an energy transf o r m i n g c o m p o n e n t e n a b l i n g establishment o f i o n gradients w i t h a higher i n f o r m a t i o n content a n d the a b i l i t y t o transfer i n f o r m a t i o n over l o n g distances [191]. E l u c i d a t i o n and comparison o f the a m i n o acid s é q u e n c e o f membrane channels. plasma membrane a n d m i t o c h o n d r i a l ATPases w i l l establish the T a m i l y tree' o f ( N a * + K * )-ATPase. I n c o r p o r a t i o n o f ( N a ' + K * ) - A T P a s e subunits o r o f selectively dissected o r m o d i f i e d enzyme fragments i n t o artificial membranes makes it then possible l o characterize the functional components o f the ( N a * - i - K * ) - A T P a s e m o l é c u l e . Acknowledgements 1 thank the Swiss N a t i o n a l Science F o u n d a t i o n for financial support (grant 3.536-0.83) and F r e d P i l l o n e l for the a r i w o r k .

350

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