388
BIOCHEMICAL SOCIETY TRANSACTIONS
polation of the H + back-flow rate to the half-time for 0,
reduction obtained from the independently measured state
3 0, uptake rate. However, the oxygen-pulse method, even
when improved by elimination of very fast H + back-flow on
the H +/H,PO,- symporter (Reynafarje et al., 1976), always
underestimates the mechanistic H + /O ratio for 3 reasons.
( I ) The half-time for 0, reduction under oxygen-pulsed conditions is much shorter than calculated from state 3 0, consumption rates. (2) H+/O flow ratios obtained from the
amount of H + at the half-time for O2 reduction, by which
time much H + back-flow has already taken place, are necessarily much lower than those obtained at level flow. (3)
There is in fact no kinetically valid way of obtaining the
H /O ratio at close to level flow by extrapolation of the total
amount of H + ejected to some point at which a given
fraction of the added O2 has been consumed, because H +
back-flow begins at time zero and increases in rate during
the course of 0, uptake as ApH increases. Only by measurement of the rates of both 0, uptake and H + ejection at conditions approaching level flow, or by measurement of both
flows and forces in conditions between level flow and static
head, can the mechanistic H + / O ratio be closely
approached.
H + / Oor q + / O ratios of 8 for succinate and approaching
12 for NADH oxidation, as we and some others have observed, raise questions as to which of the approx. 20 redox
centres of the chain are directly involved in energy transduction, and by what mechanism (whether by ligand conduction, conformational transitions, or other processes) does
this occur.
+
This work was supported by grants from the United States
Public Health Service, the National Institute of General Medical
Sciences (GM05919) and the National Cancer Institute
(CA25360).
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Proton and calcium circuits across the mitochondrial inner membrane
DAVID G. NICHOLLS, ROSALIND SNELLING
and EDUARDO RIAL
Neurochemistry Laboratory, Department of Psychiatry,
Ninewells Medical School, University of Dundee, Dundee
DDI SSY, Scotland, U . K .
The chemiosmotic theory propounded by Peter Mitchell
over 20 years ago (Mitchell, 1961) has allowed great
advances to be made in understanding not only the
mechanisms of oxidative phosphorylation, but also the
driving forces for ion transport across the mitochondrial
inner membrane. However, there are a number of complex
examples of mitochondrial ion-transport processes which
are only now being elucidated. In this paper we have
selected two different examples of interacting ion movements which have been studied in our laboratory; firstly the
mutually interdependent movements of Ca2+ and P, across
the rat liver mitochondrial inner membrane (for review see
Nicholls & Crompton, 1980; Nicholls & Akerman, 1982),
and secondly the competition between halide and protons
for transport through the 32000-M, uncoupling protein
unique to brown fat (for review, see Nicholls, 1979, 1983). In
the first case the ions move through independent pathways
but interact to form a complex in the matrix, whereas in the
second case the ions are transported by the same pathway,
but the extent to which they compete can be regulated by a
physiological modulator.
1984
606th MEETING, CORK
C a 2 + and PI movements across the inner membrane of h e r
mitochondria
The presence of independent pathways for the uptake
and release of Ca2 from the matrix (Crompton et al., 1976)
allows mitochondria to maintain a steady-state distribution
of the cation across the inner membrane regulated by the
kinetics of the uptake and efflux pathways. Although PI is
not involved stoichiometrically in either Ca2+-transport
pathway (Zoccarato & Nicholls, 1982), the anion still has
three distinct effects on C a 2 + transport: firstly it can destabilize mitochondria accumulating Ca2+ from media
lacking purine nucleotides (for review see Nicholls & Akerr a n , 1982); secondly, it can be accumulated together with
(but independently from) the cation and prevent an
alkalinization of the matrix or permanent decrease in membrane potential (Akerman, 1978); and thirdly it can form a
complex with C a 2 + in the matrix (Fig. I).
The formation of a complex will buffer the free matrix
[ C a z + ] at a value which is controlled by the solubility
product of the complex. In particular, at a constant free [PI]
the free matrix [ C a 2 + ]would be independent of the total
Ca'+ content in the matrix as long as this was adequate for
the complex to form. This prediction is consistent with the
observation that the activity of the efflux pathway is independent of matrix C a ? + load over a wide range if P, is
present in the medium (Zoccarato & Nicholls, 1982).
Secondly, the matrix free [Ca' + I would be inversely related
to the matrix free [PI](after due allowance for the stoichiometry of the complex). This is consistent with the inverse
relationship which is seen between the rate of C a Z +efflux
after addition of Ruthenium Red and the free [PI]in the
incubation (Zoccarato & Nicholls, 1982). Finally, during
this CaZ efflux the complex would dissociate, liberating
free PI ; this would normally leave the matrix in parallel to
C a 2 + via the phosphate carrier. An inhibition of C a 2 +
efflux by N-ethylmaleimide has been reported (Siliprandi et
al., 1978) and interpreted as indicating a direct involvement
of PI in C a ? + efflux. However, there is a simpler
explanation : inhibition of the independent P, carrier could
+
+
4H'
2P
Fig. I . Pathways of Ca'+ and P, transport across the
mitochondria1 inner membrane
For simplicity, phosphate is represented as P I - , i.e.
HzPO,-. Abbreviations: Resp. Ch, respiratory chain; P,
Car., phosphate carrier; Ca Uni., calcium uniporter. The
open arrows linked by a dotted line represent the reciprocal
effects of P, and Ca'+ concentrations on the other owing to
the presence of the complex.
VOl. 12
389
also inhibit CaZt efflux indirectly, by leading to an accumulation in the matrix of PIliberated from the complex but prevented from leaving the matrix. This would lower free
matrix [Ca2+ I according to the solubility product of the
complex and thus slow down further Ca" efflux.
Although there is still controversy concerning the free
matrix Ca2+ concentration, the free [PI]in the matrix may
readily be calculated by assuming equilibrium across the
phosphate carrier after allowing for the pH gradient. When
C a 2 + in the matrix is in excess over P,, the free matrix [PI]
can be less than 0.1% of the total concentration, attesting to
the stability of the complex (Snelling & Nicholls, 1982).
CaZ and PI are thus ions which, although driven by the
chemiosmotic proton circuit through independent pathways, show a considerable degree of interaction.
+
Proton and halide movements across the inner membrane of
brownyfat mitochondria
The 32000-M, uncoupling protein of brown-fat mitochondria (Heaton et a/., 1978) provides the energy-dissipating
mechanism which underlies the enormous thermogenic
capacity of the tissue (Nicholls, 1983; Nicholls & Locke
1984). The binding of purine nucleotides converts the high
ohmic proton conductance of the protein into one which is
low until a high potential is attained (Nicholls, 1977). Under
respiring conditions very low concentrations of nonesterified fatty acids override the inhibitory effect of the
nucleotides, making fatty acids powerful contenders for the
role of physiological modulators of the proton conductance
(Heaton & Nicholls, 1976; Locke et a/., 1982a,b).
Fatty acids do not, however, restore the ohmic conductance seen in the absence of nucleotide, but rather lower the
break-point potential at which the 'non-ohmic' conductance
increase is seen to a value that is no longer sufficient for
respiratory control (E. Rial & D. G . Nicholls, unpublished
work). The low diffusion potentials generated during the
swelling of non-respiring mitochondria are below the breakpoint even after this has been lowered by fatty acids. Thus,
in contrast with the respiring condition, fatty acids cannot
override the low permeability state which non-respiring
mitochondria exhibit after binding nucleotide (E. Rial & D.
G . Nicholls, unpublished work).
In addition to its proton-conducting activity, the 32000M, protein provides a pathway for halide ions which can be
inhibited by the same range of nucleotides as for proton
permeability (Nicholls & Lindberg, 1973; Nicholls, 1974).
Paradoxically, this halide conductance is not affected by the
addition or removal of fatty acids (Nicholls & Lindberg,
1974). That chloride and protons actually compete for the
same pathway can be seen from experiments, for example
swelling in KCI in the presence of nigericin (Nicholls,
1974), in which a decreased light-scattering requires the
simultaneous entry of protons (or exit of hydroxyl ions) and
entry of chloride. It is found that the two ions interfere with
each other, since swelling is much slower than when only
protons (potassium acetate plus valinomycin) or chloride
(KCI plus valinomycin) permeate through the protein.
The low swelling rate in KCI plus nigericin can be enhanced if an independent pathway for protons, i.e. a synthetic proton translocator, is provided (Nicholls & Lindberg, 1973). However, fatty acids themselves also enhance
the swelling rate in KCI plus nigericin (Nicholls & Lindberg, 1973).
We are thus faced with the following paradox:
( I ) halide ions and protons (or hydoxyl ions) compete for
the same pathway through the membrane;
( 2 ) this pathway is associated with the 32000-M, protein,
since it is responsive to purine nucleotides;
(3) fatty acids act at the 32000-M, protein to affect its
proton conductance;
390
BIOCHEMICAL SOCIETY TRANSACTIONS
+
H'
OH or Cl
Fig. 2. Schematic model for the regulation of the 32000-Mr uncoupling protein from
brown-fat mitochondria
( a ) In the absence of purine nucleotides the gate (G) is open while O H - and CIcompete to pass through the ohmic anion channel (AC). (b) Addition of purine
nucleotide closes the gate : conductance is low until bulk-phase potential rises
sufficiently for the field across the gate to induce ions to force their way through the
gate. (c) Further addition of fatty acid allows anion channel to be bypassed by
protons (but not by Cl- ). Proportion of bulk-phase potential concentrated across
gate increases; thus protons can force their way through the gate at a lower bulkphase potential. Graphs give schematic proton current (I)proton electrochemical
potential ( V ) relationships found experimentally (Nicholls, 1977; E. Rial & D. G.
Nicholls, unpublished work).
(4) fatty acids de-couple the competition between protons and chloride;
(5) fatty acids, however, have no effect on the chloride
permeability by themselves.
The model depicted in Fig. 2 reconciles these data. It is
proposed that the 32000-M, protein has two components, a
nucleotide-sensitive gate and an anion-selective transmembrane channel. The channel would not allow the simultaneous passage of protons and chloride ions in opposite
directions, and so would not allow swelling to occur in KCl
plus nigericin. Both chloride and protons (or hydroxyl ions)
could be regulated by the gate, which on binding nucleotide
would convert its conductance characteristics from ohmic
to non-ohmic. Fatty acids would enable protons (but not
chloride) to bypass the anion-selective channel (but not the
gate). This increased permeability of the transmembrane
portion of the protein would increase the field experienced
by the gate, which would then require less bulk-phase
proton electrochemical potential to achieve the local field
required to increase its conductance non-ohmically.
Crompton, M., Capano, M. & Carafoli, E. (1976) Eur. J. Biochem.
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Mitchell, P. (1961) Nature (London) 191, 423427
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Nicholls, D. G. (1979) Biochim. Biophys. Acra 549, 1-22
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Nicholls, D. G. & Akerman, K. E. 0.(1982) Biochim. Biophys. Acra
683,57-88
Nicholls, D. G. & Crompton, M. (1980) FEBS L P r f . 111, 261-268
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Nicholls, D. G. & Locke, R. M. (1984) Physiol. Rev. in the press
Siliprandi, D., Toninello, A,, Zoccarato, F., Rugolo, M. &
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Snelling, R. & Nicholls, D. G. (1982) Shorr Rep. Eur. Bioenerg.
Con$ 2nd 495496
Characteristics of the uncoupling protein from brown-fat mitochondria
M. KLINGENBERG
lnstitute tor Physical Biochemistry, University of Munich.
Goethestrasse 33, 8000 Munich 2, Federal Republic of
Germany
The inner membrane from brown-fat mitochondria contains in large abundance an integral membrane protein
Abbreviations used: DAN, dirnethylaminonaphthoyl: DCCD,
dicyclohexylcarbodi-imide;SDS, sodium dodecyl sulphate.
which is thought to be instrumental for the thermogenesis of
brown adipose tissue (Nicholls, 1979). It has been suggested
that this protein forms a channel for H +,thus short-circuiting the H + efflux generated by the respiratory chain. As
a result H +would bypass ATP synthesis and instead all the
oxidative energy would be converted into heat. The main
argument for the identitification of this protein with a H +
channel comes comes from the inhibition in mitochondria
of the uncoupling action by purine nucleotides, and the presumption, based on photoaffinity labelling, that an SDS gel
1984