Role of Water in Proton Conductance across Model and Biological

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch003

3

D.

Role of Water in Proton Conductance across Model and Biological Membranes W.

Deamer

and M. Akeson

1

2

Department of Zoology, University of California, Davis, CA 95616 Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD 20892 1

2

Proton conductance across model and biological membranes can be understood in terms of proton translocation along chains of hydrogen— bonded water molecules. This translocation mechanism accounts for the unexpectedly high permeability of lipid bilayers to proton flux, which seems to occur through rare transient defects in the bilayer barrier. The nature of the proton-conducting channel of the F F adenosine 5'-triphosphate (ATP) synthase is unknown, but may use a similar translocation mechanism. We have tested the gramicidin chan nel as a model of such proton conductance. The channel consists of a single chain of hydrogen-bonded water molecules, and its proton conductance at saturation is near 140 ρA, or 10 H / s. Assuming that an adequate supply of protons is made available to the putative channel, this rate easily supports proton transport requirements of the F F ATP synthase during ATP synthesis. However, at neutral ρH ranges diffusion of free protons probably could not maintain an adequate supply at the channel mouth. Other sources of protons must therefore be postulated. 1

9

1

T H E

0

+

0

NATURE O F T H E

PERMEABILITY BARRIER A N D T H E BASIC M E C H A N I S M

of

ion permeation are understood only i n the most general sense even though the first measurements o f ionic flux across l i p i d bilayer membranes w e r e c o n d u c t e d 25 years ago. Establishing a permeation mechanism is difficult because the fluid l i p i d bilayer is described i n terms o f average motions o f 0065-2393/94/0235-0041$08.00/0 © 1994 American Chemical Society

In Biomembrane Electrochemistry; Blank, M., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

42

BIOMEMBRANE ELECTROCHEMISTRY

many l i p i d hydrocarbon chains. T h i s description makes it difficult to deduce the conformation or activity o f a l i p i d molecule as it interacts w i t h an i o n , for instance, the permeation o f a single potassium i o n . T h e flux o f potassium ions across a liposome m e m b r a n e is readily measured, either as radioactively labeled species or w i t h a potassium-sensitive electrode. T h e flux is s l o w — about 10 "

9

the rate o f water p e r m e a t i o n d o w n equivalent g r a d i e n t s — b u t


nonetheless occurs. W h a t happens w h e n a potassium i o n approaches bilayer? Is the bilayer indifferent, o r does it b e c o m e

the

d e f o r m e d b y the

immense electrostatic energy associated w i t h the ion? H o w does the i o n enter the bilayer phase? D o e s it dissolve i n some sense, i n a manner analogous to a permeating water molecule, or does it require a substantial transmembrane defect before it can find its way across? W e were forced to address such questions

10 years ago w h e n w e

observed that p H gradients across liposome membranes decayed at rates m u c h greater than expected f r o m o u r knowledge o f other cation p e r m e a t i o n rates (J).

F o r example, a potassium i o n gradient has a decay half-time

measured i n hours, whereas an u n b u f f e r e d hydrogen i o n gradient i n the same system decays i n less than a second i f counterion current is not l i m i t i n g . T h e apparent discrepancy between p r o t o n a n d potassium permeation rates has intrinsic interest, but also has more general implications because the genera tion a n d maintenance o f hydrogen i o n gradients are central

bioenergetic

events i n all cells. W h a t u n d e r l y i n g mechanism might account for the vastly greater p e r m e ability o f l i p i d bilayer membranes to protons? T h e r e is no obvious difference between solvated forms o f potassium a n d hydrogen ions. B o t h w o u l d be " s e e n " b y the m e m b r a n e as a positive charge s u r r o u n d e d b y several water molecules. H o w e v e r , there are important differences i n the way a hydrogen i o n might be translocated across a m e m b r a n e . First, u n l i k e any other cation, a hydrogen i o n has an equivalent anion; that is, translocation o f a hydroxide i o n across a m e m b r a n e i n one direction is essentially indistinguishable f r o m the translocation o f a hydrogen i o n i n the other direction. Second, hydrogen i o n equivalents have the capacity to move along chains o f hydrogen bonds, as exemplified b y protonic conductance i n ice ( 2 , 3). I n l i q u i d water as w e l l , the ionic m o b i l i t y o f protons is about seven times that o f other cations because o f hydrogen i o n diffusion through transient clusters o f h y d r o g e n - b o n d e d water molecules. T h e u n i q u e ability o f hydrogen ions to move along h y d r o g e n - b o n d e d chains suggested a possible flux m e c h a n i s m that w o u l d differentiate between the permeation o f protons a n d other cations. Perhaps ions do not " d i s s o l v e " i n the bilayer to cross the m e m b r a n e . Instead, transient hydrated defects may be p r o d u c e d b y t h e r m a l fluctuations i n the l i p i d , a n d ions c o u l d t h e n cross the m e m b r a n e barrier b y diffusion t h r o u g h the defects. I f water molecules i n the defects are associated b y hydrogen b o n d i n g , protons c o u l d cross the


3.

DEAMER AND AKESON

43

Role of Water in Proton Conductance


defect not by diffusion, as all other ions must, but instead b y h y d r o g e n - b o n d exchange along the associated water molecules. T h e concept o f transient hydrated defects i n l i p i d bilayers w i t h h i g h selectivity for protons suggests a second possibility: If there were some way to produce a relatively l o n g - l i v e d c h a i n o f h y d r o g e n - b o n d e d water molecules across a membrane, the result w o u l d be a p r o t o n - c o n d u c t i n g channel. S u c h a channel w o u l d have important implications for the function o f certain b i o l o g i cal membranes. F o r example, the F subunit o f c o u p l i n g membranes directs protons i n such a way that previously synthesized adenosine 5'-triphosphate ( A T P ) is released f r o m b i n d i n g sites o n the F subunit. W e can ask h o w protons move t h r o u g h the F subunit: D o they diffuse t h r o u g h a channel b y a process resembling that o f other i o n channel conductance? A n alternative is that p r o t o n transfer occurs along h y d r o g e n - b o n d e d chains o f water w i t h i n F . 0

x

0

0

I n the discussion to follow, w e w i l l address the following questions: 1. W h a t is the mechanism o f the p r o t o n c o n d u c t i o n across l i p i d bilayers? 2. W h a t are the p r o t o n - c o n d u c t i n g properties o f k n o w n hydro g e n - b o n d e d chains? 3. C a n w e relate these properties to models for p r o t o n conduc tance b y the F subunit? 0

Characteristics of Proton Flux across Bilayers P r o t o n flux across l i p i d bilayers can be measured by a variety o f techniques. F o r example, a b u f f e r e d p H gradient can be established across liposome membranes, a n d the rate o f decay o f the gradient can be m o n i t o r e d b y any o f several methods. E a r l y measurements were carried out b y m o n i t o r i n g p H shifts i n the external m e d i u m w i t h a glass electrode (1); later measure ments used p H - s e n s i t i v e dyes such as pyranine, carboxyfluorescein, a n d 9-aminoacridine (4-6). Cafiso a n d H u b b e l l ( 7 ) used spin labels very effec tively, a n d Perkins a n d Cafiso (8) c o n d u c t e d an extensive series o f measure ments w i t h this system. In a typical experiment, p r o t o n flux might be driven b y p l a c i n g liposomes that contain p H 8 buffer i n a solution b u f f e r e d at p H 6, so that the initial p r o t o n flux is driven b y 1 0 ~ - M protons outside a n d 1 0 ~ hydroxide ions inside. Buffers are r e q u i r e d to make the decay rate o f p H gradients suffi ciently slow so that initial rates can be conveniently estimated. It is also necessary to release p r o t o n diffusion potentials b y addition o f v a l i n o m y c i n or a permeant anion; otherwise the p r o t o n flux w i l l be l i m i t e d b y counterion flux (9). 6

6

It is not k n o w n w h e t h e r protons or hydroxide ions conduct the p r i m a r y ionic current u n d e r these conditions, a n d N i c h o l s and D e a m e r ( i ) suggested


44


the t e r m "net p r o t o n flux" to indicate this uncertainty. H o w e v e r , i f significant conductance

o c c u r r e d b y simple diffusion o f hydroxide ions across

the

bilayer, the decay o f a p H gradient w o u l d be expected to have kinetics controlled b y the permeability o f the hydroxide anion, w h i c h s h o u l d resemble that o f other monovalent anions like chloride. A s w e w i l l see, this expectation is inconsistent w i t h evidence i n h a n d , so for the purposes o f o u r discussion Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch003

w e w i l l assume that all the current is carried as protons. U n d e r these conditions, a typical measured p r o t o n flux might b e i n the range o f 1 0 "

mol/(cm

1 5

s). T o compare this value w i t h that o f potassium,

2

1 M potassium i o n (as potassium sulfate) c o u l d be t r a p p e d inside the same liposomes, a n d potassium efflux into 1 M choline sulfate c o u l d be measured w i t h a potassium-sensitive electrode. A typical result might again be i n the range o f 10 ~

1 5

mol/(cm

2

s). T h e p r o t o n permeability anomaly n o w becomes

clear: T h e same flux is measured for b o t h potassium i o n a n d protons, yet the proton flux is driven b y a concentration o f protons 6 orders o f magnitude less than the concentration o f potassium ions. Estimates o f the relative p e r m e a b i l ities o f the bilayer to protons a n d potassium using these flux data y i e l d values o f 10 ~

6

c m / s for protons a n d 1 0 ~

1 2

c m / s for potassium i o n .

A second characteristic o f p r o t o n flux makes such calculations o f p r o t o n permeability coefficients

useful only for comparisons

o f permeability at

defined p H values. Imagine i n the p r e c e d i n g experiment that we measured potassium

ion

flux

0.01-0.001-MK . +

down

10-fold

gradients:

1.0-0.1-,

0.1-0.01-,

and

T h e flux w o u l d decrease b y an order o f magnitude w i t h

each 10-fold decrease i n potassium concentration. N o w imagine that w e measured p r o t o n conductance

across l i p i d bilayers d o w n 10-fold p r o t o n

gradients: p H 6 - 7 , 7 - 8 , a n d 8 - 9 . W h e n this experiment was carried out, the unexpected result was that the flux was relatively independent o f p H ( I ) . T h i s observation was c o n f i r m e d a n d extended b y G u t k n e c h t (10), w h o measured voltage-driven p r o t o n current across planar l i p i d membranes at p H values ranging f r o m 2 to 11 and f o u n d again that current was essentially i n d e p e n dent o f p H ; current varied approximately 10-fold over 9 orders o f magnitude difference i n p r o t o n concentration. A n y mechanism proposed for the p r o t o n flux anomaly must take this observation into account, w h i c h has p r o v e n to be a surprisingly difficult task.

Modeh for Proton Conductance in Bilayers Before discussing possible mechanisms o f p r o t o n conductance, it is w o r t h introducing the more general question o f i o n flux. T h e bilayer barrier to free diffusion o f ions was first considered b y Parsegian ( I I ) ,

w h o treated

hydrocarbon phase as solvent i n w h i c h an i o n might dissolve. T h e

the

energy

requirement can be calculated i n terms o f the B o r n energy necessary to move


3.

DEAMER AND AKESON

45


a monovalent cation f r o m water (dielectric constant = 80) into the hydrocar b o n phase o f a l i p i d bilayer (dielectric constant = 2). Parsegian showed that the energy was i n the range o f 40 k c a l / m o l for a typical ionic solute like sodium. This substantial energy requirement helps us to understand w h y a l i p i d bilayer 5 n m thick represents such an effective barrier to ionic diffusion. T h e Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch003

barrier can be overcome by p r o v i d i n g a hydrated channel, so that a c o n t i n u ous h i g h dielectric pathway exists across the bilayer. F o r example, Parsegian showed that a hydrated 0.5-nm-diameter " p o r e " r e d u c e d the B o r n energy barrier to 6.7 kcal. T h e g r a m i c i d i n channel, a classic example o f such a pore, contains a single chain of water molecules that permits ions to diffuse w i t h greatly r e d u c e d energy requirements. W e can n o w ask w h e t h e r the B o r n energy calculation can predict the experimental measurement o f an actual i o n flux. H a u s e r et al. (12)

trapped

radioactive s o d i u m i o n i n sonicated liposome preparations [now referred to as small unilamellar vesicles ( S U V ) ] and measured the s o d i u m i o n efflux over periods u p to a m o n t h . T h e measured rates were then c o m p a r e d w i t h theoretical rates p r e d i c t e d f r o m B o r n energy considerations. T h e unexpected result was that the measured permeability constant was 3 orders o f magnitude greater than the calculated value. T o explain this discrepancy, H a u s e r et al. proposed that s o d i u m ions leaked out, not b y dissolving i n the bilayer phase and diffusing across, but instead by leaking through some very substantial defect. O u r laboratory (13) earlier measurements

a n d others (14)

have c o n f i r m e d a n d extended the

o f cation permeability across liposome

[large unilamellar vesicles ( L U V ) ] a n d values i n the range o f 10 ~

membranes 1 2

cm/s have

b e e n reported. These values are m u c h greater than p r e d i c t e d f r o m B o r n energy considerations, w h i c h is again consistent w i t h the presence o f tran sient defects i n l i p i d bilayers. S u c h defects n e e d not be c o m m o n events to account for i o n permeation rates. O n e can readily calculate f r o m the mea sured flux o f s o d i u m , potassium, a n d protons ( 1 0 ~

1 5

mol)/(cm

2

s) that one

ion w i l l , o n average, pass t h r o u g h the area o c c u p i e d b y a single p h o s p h o l i p i d (0.7 n m ) every two days. B y contrast, several thousand water molecules pass 2

through the same area every second! P r o t o n permeability can n o w be c o m p a r e d w i t h the permeability of other cations i n l i p i d bilayer systems, specifically liposomes (Table I) (15-21).

First,

it is important to note that the intrinsic p r o t o n permeability o f a given l i p i d bilayer depends o n its physical state (gel or fluid) a n d the size o f the vesicles. T h i s dependence means that the p r o t o n permeability o f l i p i d bilayers can vary b y as m u c h as 3 orders o f magnitude w h e n small vesicles composed o f relatively saturated l i p i d ( P = 10 "

7

cm/s) are c o m p a r e d to large vesicles

composed of highly unsaturated l i p i d (P = 1 0 "

4

cm/s). T h e permeability is

not strongly dependent o n the l i p i d h e a d group.


46


Table I. Comparison of Permeabilities of Protons and Other Ions in Model and Biological Membranes Reference

Ρ (cm/s)

Membrane

0


Protons Large vesicles (LUV) EggPC:PA9:l Mixed plant P L Egg P C Synthetic D A P C Synthetic D M P A (T < T ) (T > TJ Small vesicles (SUV) Egg P C Planar lipid membranes Bacterial P E Diphytanoyi P C Biological membranes Sarcoplasmic reticulum Muscle sarcolemma Mitochondria Brush border membranes m

SUV, egg P C , N a L U V , egg P C , N a SUV, egg P C , Ο Γ

1.4 1.4 0.7 1.8

ΜΓ ΗΓ 10" 10 10" 10" Χ Χ Χ Χ

+

4 5

- 5

5

3

5.9 Χ 1 0 " 10" 4 Χ 10" 10 HT 10"

6

- 3

3 3

5 Χ 10" 1 0

-14

ΙΟ" ΙΟ"

1 2 1 1

1 15 8 8 16 16 8

7

5

Other Ions +

4

3

10 10 17 18 1, 19 20 12 14 21

Abbreviations: PC, phosphatidylcholine; PA, phosphatide acid; PL, phospholipid; DAPC, diarachidonylphosphatidylcholine; DMPA, dimyristoylphosphatidic acid; PE, phosphatidylethanolamine.

a

T h e permeability o f liposomes to other cations also seems to d e p e n d o n the size o f the vesicle: I n small vesicles, s o d i u m permeability ranges a r o u n d 10" c m / s , whereas i n large vesicles, s o d i u m permeability is about 100-fold greater. T h e permeability o f l i p i d bilayers to monovalent anions like chloride is consistently higher; typical values are i n the range o f 1 0 " cm/s. The question o f greater anion permeability has b e e n addressed b y F l e w e U i n g a n d H u b b e l l (22), w h o c o n c l u d e d that permeability is i n part a function o f a dipole potential at the bilayer surface that favors permeation o f anions. 1 4

1 0

Several estimates o f p r o t o n permeability o f biological membranes have b e e n made. These estimates are generally greater than the permeability o f l i p i d bilayers, as might b e expected i f the presence o f integral proteins produces defects i n the bilayer barrier that p e r m i t substantial p r o t o n leakage. H o w e v e r , even though the permeability to protons is h i g h , the concentration o f protons i n a typical biological system is so l o w (0.1 μ Μ ) that significant p r o t o n currents do not occur. F o r instance, M i t c h e l l a n d M o y l e (23) showed that the p r o t o n conductance (not permeability) o f m i t o c h o n d r i a l membranes was n o greater than that o f other cations. T h e conclusion is that a c o u p l i n g


3.

DEAMER AND AKESON


47

m e m b r a n e provides a sufficient barrier to p r o t o n flux follows, even though it may be 6 orders o f magnitude m o r e permeable to protons than to other ions

ω. W e can n o w summarize o u r conclusions regarding p r o t o n permeation o f l i p i d bilayers: Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch003

1. L i p i d bilayers have an anomalously h i g h permeability to p r o tons relative to other monovalent cations. T h e h i g h permeability, however, is balanced b y the very l o w concentration that drives p r o t o n flux i n typical membranes, so the conductance is l o w . T h i s situation permits c o u p l i n g m e m branes like those o f m i t o c h o n d r i a to maintain p r o t o n gradients even though p r o t o n permeability is h i g h . T h e p r o t o n permeability is affected b y l i p i d composition a n d vesicle size over about 3 orders o f magnitude. P r o t o n flux across bilayers has the u n i q u e property that it is independent o f p r o t o n concentration. P r o t o n flux is linearly proportional to the magnitude o f the concentration gradient d r i v i n g the flux. T h e conductance mechanism is understandable i n terms o f transient hydrated defects i n the bilayer that allow protons to translocate along h y d r o g e n - b o n d e d water chains. O t h e r cations are able to permeate through such defects, but b y diffusion, rather than h y d r o g e n - b o n d translocation. I f the defects are short-lived, the h i g h relative permeability o f protons c o u l d , therefore, b e accounted for by protons finding their way through a rare transient defect, whereas more slowly diffusing cations c o u l d not.

Proton Channels in Biological Membranes E l e c t r o c h e m i c a l p r o t o n gradients are central to many bioenergetic processes, particularly energy c o u p l i n g . T o produce such gradients, c o u p l i n g membranes contain p r o t o n " p u m p s " that are activated b y A T P , electron transport, or fight as energy sources. Examples include bacteriorhodopsin; the p r o t o n A T P a s e s o f membranes such as lyposomes, gastric mucosa, a n d secretory vesicles; the p r o t o n p u m p s c o u p l e d to electron transport enzymes such as cytochrome oxidase; a n d the F ^ A T P synthase o f c o u p l i n g membranes. S u c h p r o t o n p u m p s must have some means to translocate protons across l i p i d bilayers, and the bilayer must i n t u r n provide a sufficient barrier to p r o t o n leakage, as previously discussed. I n the second p o r t i o n o f this review w e w i l l focus o n the F subunit. 0

American Chemical Society Library 1155 16th St..Blank, N.W.M., et al.; In Biomembrane Electrochemistry; Advances in Chemistry; American Washington, Chemical O.C. Society: 20036 Washington, DC, 1994.

48


T h e F F A T P synthases p r o d u c e A T P at the expense o f electrochemical p r o t o n gradients generated b y the electron transport systems i n c o u p l i n g membranes. T h e F F structures are similar i n bacteria, chloroplasts, a n d mitochondria (24), a n d it is likely that the p r o t o n flux mechanism is essen tially the same i n all three membranes. Some p r i m a r y characteristics o f p r o t o n flux i n A T P synthases are a 1200 H / s current requirement (25) a n d H / N a = 1 0 selectivity (26) for the C F channel. C o n s e r v e d amino acids r e q u i r e d for p r o t o n permeation are a subunits A r g , H i s , a n d S e r (27) a n d c subunit A s p (28). 1

0

X

0

+


+

+

7

0

2 1 0

2 4 5

2 0 6

6 1

T h e r e are three alternative mechanisms b y w h i c h protons might cross the F subunits d u r i n g A T P synthesis. O n e possibility is that the protons pass individually through a channel b y a mechanism similar to the diffusion o f other cations t h r o u g h k n o w n transmembrane channels. H o w e v e r , true d i f f u sion o f individual protons through a classical i o n channel seems unlikely. E v e n i n a disordered environment like water, p r o t o n m o b i l i t y occurs largely by hydrogen b o n d exchange; diffusion is only a m i n o r component. T h i s situation suggests that some f o r m o f translocation along h y d r o g e n - b o n d e d chains is plausible. 0

T h e earliest suggestion that protons may be c o n d u c t e d b y a h o p p i n g mechanism along amino a c i d residues o f m e m b r a n e proteins was the " p r o t o n w i r e " concept o f N a g l e a n d M o r o w i t z ( 2 9 ) . C o x et al. (30) a n d Senior (24) proposed models i n w h i c h a network o f such residues is present along the aligned surfaces o f α-helices i n the F subunit. A l t h o u g h this is an attractive hypothesis, no such w i r e - l i k e arrangements o f amino acids have yet b e e n detected. F u r t h e r m o r e , obligatory p r o t o n conductance along wires o f amino acid residues is inconsistent w i t h evidence that N a currents can also drive A T P synthesis (31, 32). S o d i u m ions w o u l d not be expected to move w i t h i n a h y d r o g e n - b o n d e d chain o f amino a c i d residues b y the same h y d r o g e n - b o n d exchange mechanism available to protons. 0

+

T h e t h i r d alternative is p r o t o n exchange along h y d r o g e n - b o n d e d water molecules ( 3 3 - 3 5 ) . I n bacteriorhodopsin, for example, a recent structural m o d e l at 3.5-Â resolution strongly suggests that water molecules f o r m a narrow channel a n d are i n v o l v e d i n p r o t o n delivery to the chromophore (36). T h e remainder o f this review w i l l discuss chains of h y d r o g e n - b o n d e d water molecules as potential p r o t o n translocators a n d describe some initial tests o f the concept. T h e first question concerns the physical nature o f such a channel i n the F subunit. A useful m o d e l is p r o v i d e d b y the w o r k o f L e a r et al. (37, 38), w h o f o u n d that synthetic s e r i n e - l e u c i n e peptides f o r m i o n - c o n d u c t i n g chan nels i n planar l i p i d membranes. T h e channels apparently are p r o d u c e d w h e n α-helical configurations o f the peptides f o r m clusters w i t h i n the bilayer. F o r instance, w h e n a 21 residue peptide H N — ( L S S L L S L ) — C O N H was incorporated into a planar l i p i d bilayer membrane, channels appeared that h a d cation c o n d u c t i n g properties that r e s e m b l e d those o f the acetylcholine 0

2

3


2

3.

DEAMER AND AKESON

49


receptor. C h a n n e l lifetimes i n 0 . 5 - M K C l ranged a r o u n d 5 ms, w i t h conduc tances o f 70 p S (4.2 Χ 1 0 ions/s) at 1 0 0 - m V potentials. Significantly, peptides w i t h one o f the serines replaced b y a leucine f o r m e d h i g h l y selective proton channels w i t h a conductance o f 120 p S a n d lifetimes i n the range o f 1 ms. C o m p u t e r m o d e l i n g suggested that the p r o t o n channel is a trimeric o r tetrameric aggregate o f α-helices, whereas the cation c o n d u c t i n g channel is hexameric o r larger. Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch003

7

H o w might this knowledge b e a p p l i e d to p r o t o n flux t h r o u g h the F subunit? T h e F subunit o f the E. colt A T P synthase is c o m p o s e d o f approximately 10 α-helical c subunits associated w i t h one a subunit and two b subunits ( 2 7 ) . It seems reasonable to suggest as a w o r k i n g hypothesis that the c subunits encircle the a subunit. T h e i r α-helices a n d those o f the a subunit w o u l d then f o r m complexes w i t h i n the l i p i d bilayer w i t h p r o t o n c o n d u c t i n g characteristics similar to those described i n the m o d e l system o f L e a r et a l . I f the complexes are tetramers o f associated α-helices, chains o f hydrogen-bonded water molecules w o u l d b e able to translocate protons across the assembled F subunit. 0

0

0

Gramicidin as a Proton-Conducting Channel H o w c a n w e go about testing the concept that chains o f h y d r o g e n - b o n d e d water c o u l d represent the p r o t o n - c o n d u c t i n g channel o f F ? A relatively simple test concerns the rate o f p r o t o n conductance relative to A T P synthesis. U s i n g this approach, Junge a n d co-workers ( 2 5 ) measured p r o t o n flux rates through the C F channel. A f t e r correcting f o r the large fraction ( ~ 9 7 % ) o f channels that d o not conduct p r o t o n current, the unit conductance was estimated to b e 169 fS at an external p H o f 7.5, a n d 100-mV driving potential. T h i s conductance is approximately 2 X 1 0 protons p e r channel p e r second, w h i c h is more than sufficient to accommodate even the highest A T P synthesis rates (1200 H / s ) d u r i n g photophosphorylation. 0

0

5

+

W e c a n n o w ask a more specific question: C a n a single chain o f hydrogen-bonded water molecules conduct enough protons to supply mea sured rates o f A T P synthesis? T h e g r a m i c i d i n channel is able to provide some insight here. G r a m i c i d i n A produces i o n - c o n d u c t i n g channels i n l i p i d bilayers w h e n t w o g r a m i c i d i n molecules f o r m head-to-head h y d r o g e n - b o n d e d pairs that span the m e m b r a n e ( 3 9 , 40). T h e channel contains a single strand o f 1 0 - 1 2 water molecules, a n d is h i g h l y selective f o r monovalent cations. M y e r s and H a y d o n (41) first measured cation conductance characteristics o f the g r a m i c i d i n channel a n d f o u n d that p r o t o n conductance was greater than expected. It was therefore p r o p o s e d that protons were translocated b y m o v i n g along h y d r o g e n - b o n d e d water, similar to the mechanism k n o w n to occur i n ice. L e v i t t et al. (42) measured streaming potentials p r o d u c e d w h e n water is p u s h e d t h r o u g h the channel b y translocating ions. Potassium a n d s o d i u m ions


50



p r o d u c e d the expected streaming potentials, but p r o t o n flux d i d not. This observation supported the original conjecture o f M y e r s a n d H a y d o n that protons m o v e d b y a h o p p i n g mechanism along h y d r o g e n - b o n d e d water molecules i n the channel, rather than b y diffusion. T h e g r a m i c i d i n channel thus offers a m o d e l system for investigating properties o f p r o t o n conductance along h y d r o g e n - b o n d e d waters. W e can n o w address the question o f the m a x i m u m rate at w h i c h a single strand o f h y d r o g e n - b o n d e d water molecules can transport protons. I f we find that u n d e r no conditions are protons transported sufficiently fast to account for A T P synthesis b y F ^ Q , then w e must consider alternative conductance mechanisms. W e can also ask h o w the p r o t o n selectivity o f the g r a m i c i d i n channel compares w i t h that measured i n the F subunit. I f the selectivities are similar, w e can draw some conclusions about the nature o f the F channel, but i f the selectivities are vastly different, w e w i l l n e e d to consider what properties o f the F channel might p r o d u c e such a difference. 0

0

0

T h e experiments described i n the following text were carried out o n g r a m i c i d i n channels i n a glycerol monooleate-cholesterol m e m b r a n e (43). C h a n n e l o p e n times o f about 1 s were typically observed, similar to those first described b y H l a d k y a n d H a y d o n (26). W e c o m p a r e d single channel p r o t o n currents u n d e r several conditions, i n c l u d i n g varying the potential d r i v i n g the current a n d varying the concentration o f protons ( i n the f o r m o f H C l f r o m 0.01 to 7 M ) . O u r results are s u m m a r i z e d as follows: • Single channel currents: 1.4 p A i n 0.01 M H C l ; 6.0 p A i n 0.1 M H C l ; 45 p A i n 1 M H C l • Saturation current: 140 p A i n 4 M H C l or 1 0 second p e r channel

9

protons p e r

• Activation energy: 20 ± 3 k j / m o l W h e n p r o t o n currents through g r a m i c i d i n single channels were c o m p a r e d i n H C l solutions o f varying concentration, w e f o u n d three distinct conductance regimes: a shoulder at 0.01 M H C l , a linear relationship between 0.1 a n d 2.0 M H C l , a n d saturation above 4 M . T h e saturation o f the channel appears to be due to a rate-hmiting property o f the channel itself, rather than some property o f the b u l k phase i n supplying protons to the channel. T h e saturating current i n 4 M H C l is near 140 p A , equivalent to 1 0 protons per second p e r channel. T h i s is 1 0 times faster than the p r o t o n transfer rate r e q u i r e d to A T P synthesis a n d about 1 0 times faster than the m a x i m u m protonic current i n C F (25). 9

6

3

0

W e conclude that a chain o f h y d r o g e n - b o n d e d water molecules is able to translocate protons at a rate sufficient for measured A T P synthesis i f the p r o t o n supply f r o m the b u l k phase is adequate. I n the physiological p H range, the question of p r o t o n supply to a putative p r o t o n channel becomes


3.

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central. O u r current-voltage data show that p r o t o n flux i n the g r a m i c i d i n channel is l i m i t e d by supply f r o m the b u l k phase at p H ranges higher than 2 (43). P r o t o n supply to the F channel f r o m the thylakoid l u m e n ( p H 5) a n d f r o m the cytosol bathing the m i t o c h o n d r i a l m e m b r a n e ( p H 7.5) w o u l d , therefore, l i m i t p r o t o n flux. W o u l d this supply be adequate to account for measured A T P synthesis rates? T h e p r o t o n motive force i n chloroplasts a n d mitochondria is about 200 m V . A t p H 2 a n d 2 0 0 - m V a p p l i e d potential, p r o t o n current through a g r a m i c i d i n channel is 1.1 p A or 6.9 Χ 1 0 H p e r channel per second. T h e p r o t o n current i n a population o f g r a m i c i d i n channels appears to decrease linearly w i t h p H b e l o w 2 (44). Therefore, at p H 5 (the p H o f the chloroplast thylakoid l u m e n ; 45), p r o t o n flux through the grami c i d i n channel w o u l d be 6.9 X 1 0 H p e r channel p e r second. I f w e assume that the g r a m i c i d i n channel is a plausible m o d e l for p r o t o n conductance i n F , this value is sufficient for the m a x i m u m rate o f A T P synthesis i n C F Q C F ! o f intact chloroplasts.


0

6

3

+

+

0

A n adequate p r o t o n supply to an F subunit channel i n the m i t c h o n d r i a l A T P synthase is less certain. U s i n g the same logic as before, p r o t o n supply f r o m the cytosol at p H 7.5 w o u l d be only 20 H p e r channel per second. T h i s discrepancy might be overcome b y a m u c h w i d e r channel m o u t h , a slower rate o f A T P synthesis p e r enzyme, or some additional mechanism b y w h i c h protons are s u p p l i e d to the m i t o c h o n d r i a l A T P synthase. O n e possibil ity is that i n mitochondria, where A T P synthesis (and therefore p r o t o n flux) is driven b y a m e m b r a n e potential, hydrolysis o f water at the channel m o u t h c o u l d be a major source for protons. Kasianowicz et al. (46) f o u n d it necessary to invoke this possibility to account for the observed rates o f protonophore-mediated p r o t o n conductance across l i p i d bilayers. 0

+

T h e last question to be considered here is whether h y d r o g e n - b o n d e d water molecules account for the extreme p r o t o n selectivity ( 1 0 ) observed i n F . First, it is important to note that the p r o t o n selectivity observed b y L i l l et al. (25) i n C F is not due to an anomalously h i g h p r o t o n conductance, b u t rather to the apparent failure o f N a or K to permeate the channel even at 3 0 0 - m M electrolyte concentration. C F effectively excludes all ions except protons. E x c l u s i o n o f this sort c o u l d result f r o m any o f several u n d e r l y i n g characteristics o f the conductance mechanism. F o r instance, ions larger than the diameter o f the channel might be rejected, or there may be specific b i n d i n g to a site i n the channel (an energy well) a n d failure o f that site to b i n d other ions (47, 48). Rejection o f an i o n b y size seems unlikely for the F subunit. T h a t is, such a channel must be at least 2.5 Â i n diameter to accommodate water molecules, but t h e n w o u l d resemble g r a m i c i d i n , w h i c h does not exclude N a or K ions w i t h diameters significantly less than 2.5 Â. A p r o t o n selectivity ratio of 100 w o u l d be expected rather than 1 0 ( C F ) . W a t e r chains that extend only part way across the membrane, such as those postulated to lead to the c h r o m o p h o r e i n bacteriorhodopsin (37) are 7

0

0

+

+

0

0

+

+

7

0


52


m o r e likely to account for an extreme p r o t o n selectivity. T o permeate a channel containing a single chain o f water molecules, alkali cations must p u s h water molecules through the channel (42). I f displacement o f water molecules were prevented b y some physical property o f the channel, K or N a flux w o u l d be i n h i b i t e d . A second possibility is that amino acids w i t h i n the channel c o u l d specifically interact w i t h protons. F o r example, the amino groups o f arginine a n d histidine readily f o r m bonds w i t h protons but have very l o w affinities for alkali cations (49). Alternatively, the — N H groups a n d carboxylates i n F c o u l d coordinate w i t h hydrated protons a n d select against alkali cations as do certain c r o w n ethers (33).


+

+

2

0

W e can n o w summarize o u r results w i t h the g r a m i c i d i n channel a n d relate t h e m to the F subunit: 0

1. T h e p r o t o n conductance o f the g r a m i c i d i n channel has at least three rate-fimiting conditions that d e p e n d o n p r o t o n concen tration. I n the neutral p H range, the rate-limiting step appears to be p r o t o n p r o d u c t i o n b y hydrolysis o f water at the channel m o u t h . A t moderate p r o t o n concentrations ( p H 2 - 5 ) conduc tance is l i m i t e d b y p r o t o n diffusion to the channel. A t h i g h p r o t o n concentrations ( p H 0 - 2 ) conductance is l i m i t e d b y translocation along h y d r o g e n - b o n d e d water chains i n the chan nel. 2. T h e g r a m i c i d i n channel conductance saturates at 140 p A (530 pS) of protonic current i n 4 - M H C l . 3. T h e g r a m i c i d i n channel was tested as a m o d e l for p r o t o n conductance i n the F subunit u n d e r the assumptions that the channel resembled those channels p r o d u c e d b y synthetic a helical peptides i n l i p i d bilayers a n d that p r o t o n translocation o c c u r r e d along chains o f h y d r o g e n - b o n d e d water molecules. T h e measured p r o t o n conductance at saturation is more than sufficient to transport protons through the F subunit i n quan tities that w o u l d support k n o w n A T P synthesis rates. A t physio logical p H ranges the rate-limiting step appears to be p r o t o n diffusion to the channel, rather than the channel itself. F i n a l l y , the F channel must be m u c h m o r e restrictive than g r a m i c i d i n to alkali cation permeation due to either a partial water strand or to selectivity at an internal b i n d i n g site. 0

0

0

References 1. Nichols, J. W.; Deamer, D. W. Proc. Natl. Acad. Sci. U.S.A. 1980, 77 2038-2042. 2. Eigen, M.; DeMaeyer, L. Proc. R. Soc. London Ser. A 1958, 247, 505-533.



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3. Nagle, J. F.; Tristram-Nagle, S. J. Memb. Biol. 1983, 74, 1-14. 4. Nichols, J. W.; H i l l , M. W.; Bangham, A. D.; Deamer, D. W. Biochim. Biophys. Acta 1980, 596, 393-399. 5. Deamer, D. W.; Gutknecht, J. Methods Enzymol. 1986, 127, 471-480. 6. Clement, N. R.; Gould, J. M. Biochemistry 1981, 20, 1534-1539. 7. Cafiso, D. S.; Hubbell, W. L. Biophys. J. 1983, 44, 49-57. 8. Perkins, W. R.; Cafiso, D. S. Biochemistry 1986, 25, 2270-2276. 9. Deamer, D. W.; Nichols, J. W. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 165-168. 10. Gutknecht, J. J. Memb. Biol. 1984, 82, 105-112. 11. Parsegian, A. Nature (London) 1969, 221, 844-846. 12. Hauser, H.; Oldani, D.; Phillips, M. C. Biochemistry 1973, 12, 4507-4517. 13. Barchfeld, G.; Deamer, D. W. Biochim. Biophys. Acta 1988, 944, 40-48. 14. Mimms, L. T.; Zampighi, G.; Nozaki, Y.; Tanford, C.; Reynolds, J. A. Biochem istry 1981, 20, 833-850. 15. Rossignol, M.; Grignon, Ν. Grignon, C. Biochim. Biophys. Acta 1982, 684, 195-199. 16. Elamrani, K.; Blume, A. Biochim. Biophys. Acta 1983, 727, 22-30. 17. Meissner, G.; Young, R. C. J. Biol. Chem. 1980, 244, 6814-6820. 18. Izutsu, Κ. T. J. Physiol. (London) 1972, 221, 15-27. 19. Krishnamoorthy, G.; Hinkle, P. Biochemistry 1984, 23, 1640-1645. 20. Ives, H. E.; Verkamn, A. S. Am. J. Physiol. 1985, 248, F 7 8 - F 8 6 . 21. Papahadjopoulous, D.; N i r , S.; Ohki, S. Biochim Biophys. Acta 1972, 266, 561-583. 22. Flewelling, R. F.; Hubbell, W. L. Biophys. J. 1986, 49, 531-540. 23. Mitchell, P.; Moyle, J. Biochem. J. 1967, 104, 588-592. 24. Senior, A. E. Physiol. Rev. 1988, 177-231. 25. L i l l , H.; Engelbrecht, S.; Schonknecht, G.; Junge, W. Eur. J. Biochem. 1986, 160, 627-634. 26. Hladky, S. B.; Haydon, D. A. Biochim. Biophys. Acta 1972, 274, 294-312. 27. Fillingame, R. H.; Miller, M. J.; Fraga, D.; Girvin, M. E. Biophys. J. 1990, 57, 201a. 28. Cain, B. D.; Simoni, R. D. J. Biol. Chem. 1988, 263, 6606-6612. 29. Nagle, J. F.; Morowitz, H. J. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 298-302. 30. Cox, G. Β.; Fimmel, A. L.; Gibson, F.; Hatch, L. Biochim. Biophys. Acta 1986, 849, 62-69. 31. Laubinger, W.; Deckers-Hebestreit, G.; Altendorf, K.; Dimroth, P. Biochemistry 1990, 29, 5458-5463. 32. Boyer, P. D. Trench Biochem. Sci. 1988, 13, 5 - 7 . 33. Hoppe, J.; Sebald, W. Biochim. Biophys. Acta 1984, 768, 1-27. 34. Schulten, Z.; Schulten, K. Eur. Biophys. J. 1985, 11, 149-155. 35. Deamer, D. W.; Nichols, J. W. J. Membr. Biol. 1989, 107, 91-104. 36. Henderson, R.; Baldwin, J. M.; Ceska, T. Α.; Zemlin, F.; Beckmann, E.; Downing, K. H. J. Mol. Biol. 1990, 213, 899-929. 37. Lear, J. D.; Wasserman, Z. R.; DeGrado, W. F. Science (Washington, D.C.) 1988, 240, 1177-1181. 38. DeGrado, W. F.; Lear, J. D. Biopolymers 1990, 29, 2065-2213. 39. Cornell, B. J. Bioenerg. Biomembr. 1987, 19, 655-676. 40. Urry, D. W.; Goodall, M. C.; Glickson, J. D.; Mayers, D. F. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1907-1911. 41. Myers, V. B.; Haydon, D. A. Biochim. Biophys. Acta 1972, 274, 313-322.



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42. Levitt, D. G.; Elias S. R.; Hautman, J. M. Biochim. Biophys. Acta 1978, 512, 436-451. 43. Akeson, M.; Deamer, D. W. Biophys. J. 1991, 60, 101-109. 44. Eisenman, G.; Enos, B.; Sandblom, J.; Haggland, J. Ann. Ν.Ύ. Acad. Sci. 1980, 339, 8-20. 45. Althoff, G.; Lill, H.; Junge, W. J. Membr. Biol. 1989, 108, 263-271. 46. Kasianowicz, J.; Benz, R.; McLaughlin, S. J. Membr. Biol. 1987, 95, 73-89. 47. Tsien, R. W.; Hess, P.; McCleskey, E. W.; Rosenberg, R. L. Annu. Rev. Biophys. Biophys. Chem. 1987, 16, 265-290. 48. Eisenman, G.; Dali, J. A. Annu. Rev. Biophys. Biophys. Chem. 1987, 16, 205-226. 49. Williams, R. J. P. Annu. Rev. Biophys. Biophys. Chem. 1988, 17, 71-97. RECEIVED f o r review January 2 9 , 1 9 9 1 . ACCEPTED revised manuscript June 24, 1992.


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