Biomembrane Electrochemistry - American Chemical Society

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Downloaded by FUDAN UNIV on March 22, 2017 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch012

The Mitochondrial Voltage-Dependent Anion-Selective Channel Marco Colombini Laboratories of Cell Biology, Department of Zoology, University of Maryland, College Park, MD 20742

The mitochondrial outer membrane contains a 30-kDa protein called voltage-dependent anion-selective channel (VDAC) that forms channels with 3-nm pores through which metabolites travel between the cytoplasm and the mitochondrial spaces. Electron micrographs of two-dimensional crystals of these channels after freeze-drying, shadowing, and computer processing reveal detailed surface images of the channels. Both surfaces look very similar: most of the protein appears to be embedded in the membrane. The ion selectivity of the channel and changes in it induced by site-directed mutations fit quite well to the fixed-charge theory of Teorell. The voltage-dependent closure of VDAC at both positive and negative potentials can be modulated by polyanions, osmotic pressure, aluminum hydroxide, and a soluble mitochondrial protein called the VDAC modulator. VDAC acts as a binding site for proteins perhaps through a domain located on the membrane surface.

^ M I T O C H O N D R I A C O N T A I N T W O A Q U E O U S C O M P A R T M E N T S , one i m p e r m e a b l e

to sucrose a n d the other permeable to sucrose b u t i m p e r m e a b l e to h i g h molecular-weight polysaccharides. T h i s description was p r o p o s e d b y W e r k h e i s e r a n d Bartley ( I ) i n 1956. T h e i r conclusion that m i t o c h o n d r i a consist o f t w o membrane-enclosed compartments, one inside the other, is universally accepted today. Translocation across the i n n e r membrane is p e r f o r m e d b y a host o f specific transport systems whereas movement across the outer m e m b r a n e occurs v i a a protein, called voltage-dependent anion0065-2393/94/0235-0245$08.00/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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selective channel ( V D A C ) , that forms large aqueous pores. T h e size selectiv ity for nonelectrolytes observed b y early investigators for the sucrose-permea ble compartment (now k n o w n as the i n t e r m e m b r a n e space) c a n b e readily understood b y the presence o f these channels (2-4). V D A C channels w i t h virtually the same properties have b e e n isolated f r o m m i t o c h o n d r i a representing a l l the eukaryotic kingdoms ( 5 ) . T h e only source o f m i t o c h o n d r i a lacking V D A C channels is a yeast strain whose V D A C gene was skillfully deleted ( 6 , 7 ) . A major f u n c t i o n o f m i t o c h o n d r i a — t o provide cells w i t h adenosine 5'-triphosphate ( A T P ) p r o d u c e d b y oxidative p h o s p h o r y l a t i o n — r e q u i r e s that A T P , adenosine 5'-diphosphate ( A D P ) , inorganic phosphate (Pi), a n d metabo lic substrates cross the outer m e m b r a n e . T h e discovery (8) that V D A C channels are voltage-gated raises possibilities f o r regulation w i t h far-reaching consequences. I f the outer m e m b r a n e w e r e to b e c o m e i m p e r m e a b l e to critical metabolites, important functions such as m i t o c h o n d r i a l A T P p r o d u c tion w o u l d cease.

Structure of the VDAC Channel H i g h - r e s o l u t i o n images o f V D A C channels have b e e n obtained b y taking advantage o f the fact that V D A C channels i n the Neurospora crassa m i t o chondrial outer membranes c a n b e i n d u c e d to f o r m large two-dimensional crystals ( 9 , J O ) . E l e c t r o n micrographs o f these crystals c a n b e converted into high-resolution averaged images t h r o u g h the use o f c o m p u t e r averaging a n d filtration techniques. Negatively stained crystals allow the aqueous pore f o r m e d b y the channel to b e visualized ( F i g u r e I D ) . F r o z e n - h y d r a t e d m e t h ods have visualized the p r o t e i n regions f o r m i n g the pore (11). M o r e recently, a f r e e z e - d r y i n g - s h a d o w i n g procedure was used to visualize the surface topography o f each side o f the channel ( F i g u r e 1A a n d B ) . T h e results clearly show that V D A C forms a large aqueous pore w i t h a small amount o f p r o t e i n ; most o f the p r o t e i n is e m b e d d e d i n the m e m b r a n e ( F i g u r e 2 A a n d B ) . T h e proteins that f o r m V D A C channels have b e e n sequenced for three species, N. crassa, S. cerevisiae, a n d H. sapiens. A l l these species are probably c o m p o s e d o f 282 a m i n o acids a n d have molecular weights o f 29,848, 29,752, a n d 30,641, respectively (12-14). T h e closeness o f the molecular weights a n d amino a c i d content between proteins f r o m fungi to mammals is i n h a r m o n y w i t h the high degree o f similarity i n the properties o f V D A C f r o m many different sources ( 5 ) . T h e degree o f amino a c i d identity between the fungal sequences a n d the h u m a n is only 2 4 - 2 9 % (14), b u t the degree o f conservation is underestimated. Probes into the secondary structures o f the three c h a n n e l - f o r m i n g p r o teins reveal a h i g h degree o f conservation. T h e nature o f the channel a n d its location w i t h i n the m e m b r a n e greatly l i m i t the possible secondary structures. A cylinder f o r m e d p r i m a r i l y o f a beta-sheet w a l l was p r o p o s e d ( 6 , 15) a n d



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The Mitochondrial Channel, VDAC

247

Figure 1. Two-dimensional crystals of VDAC from N . crassa were freeze-dried and shadowed with platinum. The electron micrographs were digitized, and averaged and filtered images were obtained by computer processing. A, The average reconstructed image of filtered, freeze-dried-shadowed (45°) VDAC crystals as seen from one membrane surface. The dark areas are depressions, presumably the openings of the channels. The same crystal was viewed from the other surface of the membrane, B. The image in Β was flipped about the axis of mirror symmetry and aligned with that in A by cross-correlation, and then averaged, C. This image represents eight crystals with different shadowing directions. D, The Fourier-filtered image of a negatively stained VDAC crystal. The dark areas are the location of stain accumulation, presumably the pores of the channels. All images are at the same magnification. The bar marker = 20 nm. (Reproduced with permission from reference 19. Copyright 1991 Academic.)

supported b y site-directed mutagenesis o f V D A C f r o m yeast ( 1 6 ) . S u c h a beta-sheet wall, w h i c h separates the polar environment w i t h i n the channel f r o m the nonpolar environment o f the bilayer, w o u l d have to b e c o m p o s e d o f beta strands w i t h amino acid side chains that alternate between polar a n d nonpolar residues. T h i s w o u l d f o r m a " s i d e d " strand that is w e l l suited to f o r m the interface between these t w o different phases. A c o m p u t e r search ( 6 ) f o r such alternating patterns i n 10-amino-acid stretches (3.5 n m long; enough to span the nonpolar p o r t i o n o f the membrane) o f the p r o t e i n sequence revealed many locations ( F i g u r e 3). These locations were used to generate a m o d e l f o r the o p e n state o f V D A C that was tested b y site-directed mutations a n d f o u n d to be largely correct ( 1 6 ) . T h e location o f the amino acid stretches appropriate to f o r m the " s i d e d " beta strands is w e l l conserved



248


Figure 2. Three-dimensional representation of the averaged surface reconstructions. A and Β correspond to Figure 1A and B, respectively. Dark areas are regions of lowest elevation and white areas are those of highest elevation. (Reproduced with permission from reference 19. Copyright 1991 Academic.)

a m o n g the species ( F i g u r e 3), w h i c h indicates a very similar secondary structure ( 1 7 ) . D e s p i t e earlier indications that V D A C is a d i m e r , recent evidence strongly favors a m o n o m e r . E a r l y attempts to quantitate the amount o f p r o t e i n mass i n the two-dimensional crystals o f V D A C i n d i c a t e d that there may not b e enough mass to f o r m a d i m e r (11, 18). M o r e recently, the use o f scanning transmission electron microscopy o n the V D A C crystals allowed precise estimates o f mass p e r channel (19). T h e s e scans showed a 1:1 relation between channels a n d 3 0 - k D a polypeptide chains i f the crystal also contained 3 2 % l i p i d . Results (20) o f attempts to make h y b r i d channels b y growing cells



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Beginning Residue Number Figure 3. An evaluation of the potential of stretches of amino acids in the VDAC sequences from S. cerevisiae and N . crassa and of a homologous human sequence to form beta strands lining the walls of a water-filled pore. The hydropathy values of each group of 10 adjacent amino acids were combined as follows: Σ{ί ( — l) v(\), where v(i) is the hydropathy value of the ith amino acid. The absolute value of these sums was plotted against the number of the first amino acid in the summation. The numbers in panel Β represent the locations of the major peaks in this panel. The numbers in panels A and C refer to major peaks found in these panels that were minor or absent in panel B. (Reproduced with permission from reference 17. Copyright 1991 Academic.) 1

l+1

containing two different V D A C genes (one w i l d a n d one mutant) are support ive o f a m o n o m e l i c channel. C h a n n e l s isolated f r o m the m i t o c h o n d r i a o f such cells were either w i l d o r entirely mutant i n their properties. N o channels were observed w i t h properties intermediate between the w i l d a n d mutant phenotypes. T h u s it is likely that only one 3 0 - k D a polypeptide, w h i c h is capable o f two separate voltage-gating processes, forms a cylindrical channel that results i n a 3 - n m ( i n diameter) aqueous pore through the m e m b r a n e . F r o m the h y d r o d y n a m i c properties o f V D A C isolated f r o m rat liver, L i n d e n a n d Gellerfors (21) d e d u c e d that V D A C exists as a d i m e r i n deter-



250


gent solution. T h i s finding, a n d the observation that the rate o f channel insertion into planar membranes is linearly dependent o n the amount o f detergent-solubilized p r o t e i n a d d e d to the aqueous phase, l e d early o n to the conclusion that V D A C was a d i m e r . A reexamination o f the hydrodynamie data shows that no correction was made for the large aqueous pore o f V D A C . T h e measured sedimentation coefficient, Stokes radius, a n d partial specific v o l u m e were used to determine the effective molecular weight o f detergentsolubilized V D A C . T h e calculated value o f 171,000 for the molecular weight was corrected for detergent b i n d i n g a n d resulted i n a net value o f 61,000. A further correction for water i n the aqueous pore (3 n m i n diameter a n d 5 n m long) w o u l d y i e l d a net value o f 40,000. T i g h t l y b o u n d water and lipids c o u l d account for the difference between 40,000 a n d the actual molecular weight o f 30,000. T h u s the hydrodynamie data are consistent w i t h a m o n o m e l i c chan nel.

Channel Selectivity T h e charge selectivity o f channels that f o r m a large aqueous pore is likely to be d o m i n a t e d b y the electrostatic environment w i t h i n the pore rather than the b i n d i n g o f the passing ions to the w a l l o f the pore. Therefore, charged residues o n the w a l l s h o u l d have strong effects. T h i s expectation was verified for V D A C b y using site-directed mutations to change residues at specific locations (16). Indeed, identification o f residues fining the w a l l o f the pore was possible b y d e t e r m i n i n g whether a charge change at that location resulted i n the appropriate change i n selectivity as measured b y d e t e r m i n i n g the reversal potential i n the presence o f a K C 1 salt gradient. Greater insight into the molecular basis for selectivity i n V D A C was obtained (22) b y c o m p a r i n g the observed reversal potentials as a function o f the engineered charge change w i t h that expected f r o m the fixed-charge m e m b r a n e theory o f T e o r e l l (23). F i g u r e 4 reveals a remarkably g o o d fit between reversal potential changes due to charge changes at residues thought to be i n the pore (triangles a n d circles) a n d the theory (solid curve). C h a r g e changes at locations thought to be outside the pore (squares) h a d no significant effect o n the reversal potential. T h e data p l o t t e d as circles all contained the mutation K 1 9 E (the lysine at position 19 converted to gluta mate) that displays a stronger than expected effect o n the selectivity. O v e r a l l , the theory a n d experimental observations agree very w e l l , w h i c h indicates that the molecular basis for i o n selectivity i n V D A C can b e understood u s i n g a fairly simple theory.

Voltage Dependence E a c h V D A C c h a n n e l can close at b o t h positive a n d negative potentials ( F i g u r e 5), w h i c h indicates the presence o f two gating processes w i t h i n the



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- 2 0 «J

-4

The Mitochondrial Channel VDAC

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1

1

1

1

1

1

1

1

1

1

-3

-2

-1

0

1

2

3

4

5

6

Charge in the Pore Figure 4. The change on the reversal potential of the VDAC channels with change in the charge within the pore was calculated by applying the theory of Teorell to the VDAC channel (solid line). The charge within the pore for the wild-type channel (solid diamond) was chosen to lie close to the theoretical curve. All other points were plotted according to the measured reversal potentials and the charge change induced by the mutation. Mutations at sites determined to be within the pore by other criteria were plotted as tnangles or circles; those mutations determined to be outside the pore were plotted as squares. The salt concentration gradient and sign of the potential are indicated in Table I. Taken from the data of Feng et al. (22).

p r o t e i n . T h e most conductive ( " o p e n " ) state is t h e p r e f e r r e d conformation at l o w o r zero m e m b r a n e potential. A t h i g h fields, l o w - c o n d u c t i n g ("closed") states are o c c u p i e d . I n K C 1 solutions, the conductance o f the most frequently observed closed state is 4 0 - 5 0 % o f the conductance o f the o p e n state. H o w e v e r , there is evidence (24-26) to indicate that the closed states are i m p e r m e a b l e to the biologically important ions ( A T P a n d A D P ) . T h e fact that these closed states c a n still conduct K C 1 has a l l o w e d researchers to study the properties o f these states. T h e process o f channel closure i n V D A C must involve rather extensive structural changes. T h e effective pore diameter is r e d u c e d f r o m 3 to 1.8 n m (27, 28). T h e v o l u m e o f aqueous solution w i t h i n the pore is r e d u c e d b y about 30 n m ( 2 9 ) . T h e selectivity o f the channel, as measured b y t h e reversal potential f o r a 10-fold K C 1 gradient, reverses (4, 30) f r o m a m i l d preference f o r anions to a preference f o r cations (a change o f over 30 m V i n reversal potential). T h e slow rate o f c h a n n e l closure [from milliseconds to seconds, d e p e n d i n g o n the a p p l i e d voltage ( 2 , 8)] is consistent w i t h a n extensive conformation change. Surprisingly, the rate o f c h a n n e l o p e n i n g is 3



252


Figure 5. The voltage dependence of VDAC as illustrated by the behavior of a single VDAC channel from P . aureûa. The upper trace shows the current trace recorded under voltage-clamp conditions. The lower trace is the applied voltage. The zero current and voltage levels are indicated. A steep slope in the current indicates a high conductance; thus, the channel was in the open state. Transitions to a shallower slope are channel closures (the reverse is an opening). Time proceeded from left to right. The channel was open at low potentials and closed at both positive and negative potentials. (Reproduced with permission from reference 5. Copyright 1989 Springer-Verlag.) fast (microseconds) i f the channels have not b e e n a l l o w e d to adapt to the closed state ( 2 , 8 ) . T h e h i g h sensitivity that c a n be achieved w h e n current flowing through a planar p h o s p h o l i p i d m e m b r a n e is r e c o r d e d allows the properties o f i n d i v i d u a l channels to b e studied. I n this way, variability i n properties f r o m c h a n n e l to c h a n n e l c a n b e examined a n d multiplicities i n the n u m b e r o f states that the channel can achieve c a n b e observed. B y raising a n d l o w e r i n g the a p p l i e d voltage, a V D A C channel c a n b e i n d u c e d to o p e n a n d close. I n p r i n c i p l e , the channel may sample many different conformations, a n d these conformations c o u l d b e distinguished b y their properties such as conductance a n d i o n selectivity. I n practice, f o r V D A C the properties o f the o p e n state are quite constant whereas the properties o f the closed state are m u c h m o r e variable, w h i c h demonstrates a multiplicity o f closed states ( 3 0 ) . T h e r e are w i d e variations i n b o t h the size a n d selectivity o f the closed states. T h e variability is somewhat greater between channels than f o r the same channel, b u t not b y very m u c h . T h e variability c a n be dramatically r e d u c e d b y a d d i n g agents that increase the voltage dependence (see T a b l e I; note change i n standard deviation). T h e s e agents seem to favor a particular closed-state conformation.

Modulation of Voltage Dependence of VDAC T h e voltage-gating process o f a channel c a n b e i n f l u e n c e d i n two ways: 1. T h e switching region (i.e. the voltage range over w h i c h chan nels go f r o m p r e d o m i n a n t l y o p e n to p r e d o m i n a n t l y closed) c a n be m o v e d along the voltage axis. 2. T h e voltage dependence can b e increased o r decreased to y i e l d a narrower or w i d e r switching region.


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Table I. Single-Channel Properties with and without Konig's Polyanion Plus 0.5 Property Open channel Conductance (nS) Reversal potential (mV) Closed channel Conductance (nS) Reversal potential (mV)

pg/mL

Control

Polyanion

1.8 ± 0.1 (11) 10.0 ± 1.1 (11)

1.9 ± 0.1 (3) 10.5 ± 0.6 (3)

0.81 ± 0.17 (7) - 8.6 ± 1 . 4 (7)

1.2 ± 0.07 (3) - 22.6 ± 1 . 0 (5)

N O T E : Measurements made in 1 M KCl vs. 0.1 M KCl with 5 mM C a C l , 1 mM 2-(N-morpholine)ethanesulfonic acid, pH 5.8, on each side (sign refers to high-salt side). Values are means plus or minus standard deviation, and the number of estimates is given in parentheses.


2

M o v e m e n t of the switching region is quantitated b y measuring V , the 0

voltage at w h i c h the channels have an equal probability of b e i n g either o p e n or closed (8).

A l t e r a t i o n of the voltage dependence is quantitated b y deter

m i n i n g the parameter η—the

n u m b e r o f charges that w o u l d have to move

through the entire m e m b r a n e potential to account for the observed voltage dependence. B o t h of these mechanisms and a c o m b i n a t i o n o f the two were observed i n V D A C . A variety o f highly negatively charged polymers f r o m synthetic polyanions (such as dextran sulfate) to highly negatively charged proteins (such as pepsin) can increase the voltage dependence o f V D A C and result i n b o t h an increase

i n η a n d a decrease i n V

0

(31,

32).

Voltage-dependence

can

increase more than 20-fold ( P . M a n g a n a n d M . C o l o m b i n i , u n p u b l i s h e d results) a n d result i n large conductance changes for a fraction o f a millivolt change i n m e m b r a n e potential. Because asymmetric addition effects only one gating process (31),

the polyanion must be a d d e d to b o t h sides o f the

m e m b r a n e to influence b o t h gating processes. T h i s exquisite sensitivity can be exploited easily b y cells to detect very small changes

in

membrane

potential. T h e voltage dependence can be m o d u l a t e d i n this way by regula tion of the charge o n a p o l y m e r such as the degree o f phosphorylation o f a protein. A particularly potent polyanion that was p r o d u c e d b y Kônig et al. (33) had potent effects ( 2 7 ) at less than 1 μ g / m L . I n addition to increasing the voltage dependence o f V D A C (as was the case for the other polyanions), this polyanion also caused channels to r e m a i n closed even i n the absence o f a m e m b r a n e potential. A d d i t i o n of this polyanion to the solution o n b o t h sides o f the membrane resulted i n a m a r k e d effect o n b o t h gating

processes,

whereas addition to only one side o f the m e m b r a n e resulted i n selective stimulation of the gating process i n response to negative potentials o n the polyanion side (22). T h i s polyanion also reduces the size o f negatively stained pores o f two-dimensional crystals o f V D A C

channels, w h i c h results i n a


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reduction i n observed pore diameter to 1.7 n m (28)

that is very consistent

w i t h estimates f r o m nonelectrolyte fluxes (1.8 n m ; 27).

T h i s polyanion also is

an effective

i n h i b i t o r o f A D P a n d A T P flux across m i t o c h o n d r i a l outer

membranes

(24~26).

T h e switching region can be m o v e d along the voltage axis without affecting the steepness o f the V D A C voltage dependence b y changing the colloidal osmotic pressure o f the m e d i u m (29).

T h e effect may be enhanced

by higher molecular weight polymers ( P . S. M a n g a n a n d M . C o l o m b i n i , unpublished) o r charged polymers. I n addition, there is a synergistic effect o f polyanions a n d osmotic pressure that can result i n enormous effects ( P . S.


M a n g a n a n d M . C o l o m b i n i , unpublished). Z i m m e r b e r g a n d Parsegian p r o posed ( 2 9 )

that a c e l l c o u l d use V D A C

to m o n i t o r a n d regulate

the

concentration o f intracellular polymers. A water-soluble p r o t e i n that increases the rate a n d extent o f V D A C channel closure has b e e n identified (34)

a n d localized to the m i t o c h o n d r i a l

i n t e r m e m b r a n e space ( M . H o l d e n a n d M . C o l o m b i n i , u n p u b l i s h e d observa tions). T h i s p r o t e i n , c a l l e d the V D A C modulator, m a y b e the natural sub stance that the polyanions m i m i c , b u t its h i g h potency (operating i n the nanomolar range; 34)

indicates a m u c h m o r e specific a n d d i r e c t e d action

than s i m p l y acting as a p o l y a n i o n . L i k e Konig's polyanion, it binds to V D A C and induces m o r e complex effects. T h e importance o f the V D A C modulator is indicated b y the remarkable degree o f conservation o f the protein. N o t only can it be isolated f r o m the m i t o c h o n d r i a o f species as diverse as rat, potato, and

fungi, but the modulators isolated f r o m each species act o n

isolated f r o m any o f the species

VDAC

(35).

M i c r o m o l a r amounts o f a l u m i n u m hydroxide p r o f o u n d l y reduce voltage dependence o f V D A C (36,

37).

the

A l t h o u g h initial results w e r e consis

tent w i t h a direct neutralization o f the voltage sensor (36),

further w o r k

(30)

indicates that an indirect effect is m o r e likely. T h e presence o f a l u m i n u m hydroxide i n the compartment o n one side o f a m e m b r a n e inhibits channel closure w h e n that side is made negative. Positive potentials o n the a l u m i n u m side result i n V D A C

closure, but c h a n n e l r e o p e n i n g is i n h i b i t e d . T h i s

p h e n o m e n o n can be explained i n terms o f an a l u m i n u m hydroxide b i n d i n g site that is translocated across the m e m b r a n e

(30).

VDAC as a Site for Protein Attachment T h e images o f the freeze-dried V D A C crystals ( F i g u r e 2 A a n d B ) reveal that V D A C channels do not p r o t r u d e into the b u l k aqueous phase. Indeed, the protein that forms the rim o f the pore seems to be at or b e l o w the level o f the m e m b r a n e surface (19).

T h i s lack of protrusion was also observed i n nega

tively stained membranes ( 9 )

a n d may account for the observation that


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is quite resistant to the action o f proteases ( 9 ) . I f V D A C

lacks

domains extending into the b u l k phase, h o w c a n proteins like the V D A C modulator (34),

hexokinase (38-40),

a n d m i c r o t u b u l e associated

proteins

(41) b i n d to the channel without obstructing the i o n flow? A n u m b e r o f laboratories have reported strong evidence that hexokinase a n d other kinases (42) b i n d to m i t o c h o n d r i a b y attaching to V D A C .

(38-40)

Attempts to observe t h e effects o f hexokinase o n channel behavior have b e e n unsuccessful ( P . S. M a n g a n a n d M . C o l o m b i n i , u n p u b l i s h e d results). T h u s , i t is possible that hexokinase binds b u t does not affect channel conductance o r behavior.


O t h e r b i n d i n g agents seem to have n o effect o n channel behavior. Concanavalin A binds to rat liver V D A C i n the t r i t o n X l O O - s o l u b i l i z e d f o r m (4, 43). Antibodies against

N. crassa V D A C i n h i b i t V D A C insertion into

planar membranes a n d b i n d to V D A C crystals i n membranes, b u t d o not affect channel behavior (44).

T h e s e agents m a y interact w i t h a surface

d o m a i n o n V D A C that does not r e s p o n d to m e m b r a n e potentials. B l a c h l y - D y s o n a n d co-workers m a p p e d out regions o f the V D A C p r o t e i n that f o r m the walls o f the pore (16). M a n y o f t h e p r o t e i n loops attributed to the surface are rather short. H o w e v e r , there is a m u c h longer region that extends f r o m amino a c i d 186 to 227 that, based o n the fact that amino a c i d substitutions i n this region d o not effect c h a n n e l selectivity (16, 22), is o n the surface. T h i s region is a good candidate f o r a surface d o m a i n that proteins might b i n d to. Structural evidence f o r this surface d o m a i n may b e the elevated regions i n the freeze-dried images ( F i g u r e 2 A a n d B ) . A l t h o u g h these regions are likely to contain lipids, p r o t e i n m a y also b e present. Freeze-fracture

images

( F i g u r e 6) show elevations i n the same location as the elevations seen i n the freeze-dried images.

O n e interpretation o f the lower resolution

freeze-

fracture images is that the channels f o r m the depressions between the raised areas. I f the raised areas were just l i p i d , the fracturing process s h o u l d have r e m o v e d the t o p layer o f l i p i d , resulting i n a depression. T h e presence o f a n elevation m a y indicate that the lipids are covered b y a p r o t e i n d o m a i n that links the six channels into o n e visible particle a n d also forms a b i n d i n g site f o r proteins. F r o z e n - h y d r a t e d specimens indicate the presence o f p r o t e i n i n this region ( J J ) a n d there is evidence that cytochrome c binds to this region (45).

Summary and Conclusion T h e V D A C channel has a rather simple structure a n d yet performs a variety o f interesting tasks. T h e simple c y l i n d r i c a l channel has selectivity, t w o volt age-gating processes, a n d a b i n d i n g site (or sites) f o r enzymes a n d m o d u l a t i n g proteins. T h e h i g h degree o f conservation o f structure a n d properties i n d i -




256

Figure 6. A, Freeze-fracture electron micrograph ( Χ 200,000) of a mitochondrial outer membrane vesicle of N . crassa. The micrograph is printed with black shadows. The arrow indicates the direction of shadowing. Bar marker — 100 nm. B, Averaged image of the freeze-fractured array. Defects in the lattice of the array were corrected for by cross-correlation between unit cells and a reference alignment of the unit cells. The arrow indicates the direction of shadowing. Bar marker = 20 nm. This is unpublished data from L. Thomas and M. Colombini. C, Interpretation of the particles viewed in the freeze-fracture image as suggested by Carmen Mannella, who also provided the image. This image is an averaged projected image of a negatively stained crystalline array of channels from N . crassa. A dark line drawn around each six-channel unit may correspond to one quasihexagonal particle observed in the freeze-fractured image.


12.

COLOMBINI

The Mitochondrial Channel,

VDAC

257

cates the existence o f important physical or physiological constraints. A n understanding o f h o w this channel works a n d the physiological roles it serves is likely to be achieved i n the near future.


References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

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Biomembrane Electrochemistry - American Chemical Society

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