583rd MEETING, CAMBRIDGE
969
This work was supported by the Medical Research Council.
Boyd, G . S.,Grimwade, A. M. & Lawson, M. E. (1973)Eur. J. Biochem. 37,334-340
Dempsey, M. E. (1974) Annu. Rev. Biochem. 43,947-990
Gaylor, J. L. & Delwicke, C. V. (1976)J. Biol. Chem. 251,6638-6645
Mester, J., Robertson, D. M., Feherty, P. A. & Kellie, A. E. (1970) Biochem. J . 120, 831-836
Mitton, J. R., Scholan, N. A. &Boyd, G . S . (1971) Eur. J. Biochem. 20,569-579
Ritter, M. C. & Dempsey, M. E. (1971)J.Biol. Chem. 246,15361539
Spence, J. T. & Gaylor, J. L. (1977)J.Biol. Chem. 252,5852-5858
Wirtz, K. W. A, (1974)Biochim. Biophys. Acta 344,95-117
The Estimation of the Electrical Potential across the Inner Membrane of
Mitochondria within Intact Synaptosomes
I. D. SCOTT and D. G. NICHOLLS
Neurochemistry Laboratory, Department of Psychiatry, Ninewells Medical School,
University of Dundee, Dundee DD1 SSY, Scotland, U.K.
The isolated nerve ending, or synaptosome, represents the simplest system for the
investigation of the overall processes associated with neurotransmitter release. Synaptosomes contain mitochondria that when isolated (Lai et af., 1977; Nicholls, 1978a) are
capable of developing a high ApH+ (proton electrochemical gradient), transporting
Caz+ (Nicholls, 1978a) and lowering the extramitochondrial free Caz+ concentration
to well below 1 , u (D.
~ G. Nicholls, unpublished work). The magnitude of the mitochondrial membrane potential (Aw,,,) affects the ability of mitochondria to buffer
extramitochondrial free Ca2+concentrations (Nicholls, 19786). If A i m could be determined in intact synaptosomes the ability of the mitochondria in situ to regulate the
cytosolic Caz+could be assessed.
In this present communication we describe a technique based on methods developed
for the isolated hepatocyte (J. B. Hoek, D. G. Nicholls & J. R. Williamson, unpublished
work). Methyltriphenylphosphonium is a lipophilic cation that permeates by electrical
uniport across a variety of membranes (Skulachev, 1970). With intact synaptosomes the
considerable accumulation of the cation suggests that it is being accumulated by the
internal mitochondria as well as distributing to a Nernst equilibrium across the plasma
membrane.
If the synaptosome is treated as a two-compartment system, comprising cytosol and
mitochondrial matrix, then methyltriphenylphosphonium should achieve a Nernst
equilibrium across both the plasma membrane and the inner mitochondria1 membrane.
The total uptake of methyltriphenylphosphoniumwill thus be a function of the volumes
of the respective compartments, the plasma membrane potential (Aw,) and the mitochondrial membrane potential (Aty,,,). If TPMP+ represents methyltriphenylphosphonium and e, c and m represent respectively the external, cytosolic and matrix
comDartments. then :
[TPMP+lc- 1OA*p/60
[TPMP+],
If [TPMP+], is the experimentally determined overall concentration of methyltriphenylphosphonium in the synaptosome, and V, and V,,, are the volumes of the cytosolic
and matrix compartments respectively, then :
VOl. 7
970
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Electricalpotentials across the plasma membrane (Awp) and mitochondria1 inner
membrane (Aw,) of intact synaptosomes
Synaptosomes (final concn. 2mg of protein/ml) were incubated at 30°C at pH7.4 in
a medium containing 122m~-NaCI, 3.1 mM-KCI, 1.2m~-MgSO,, 1.3m~-CaCI,,
0.4 ~ M - K H ~ P O5 mM-NaHC03,
,,
20 mM-sodium N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate, 10mM-D-glucose, 35p~ - '~ R bC I(O.lpCi/ml), 25p~-['~C]sucrose
bromide (0.7pCilrnl) and 5 p ~ (0.5pCi/ml), 1fi~-[~H]methyltriphenylphosphonium
tetraphenylboron. Further additions were made after lOmin, and after a further 90s
the incubation was centrifuged in an Eppendorf model 5412 centrifuge. Potentials
were calculated as described in the text.
Further addition
None
OAfl~-Carbonylcyanide p-trifluoromethoxyphenylhydrazone
~O~M-KCI
[Rb+],
[Rb+].
13.1
11.4
[TPMP+],
[TPMP+],
116
17.6
5.4
54
A Y ~
(mV)
67
63
AVm
(mV)
150
80
44
153
The total synaptosomal volume ( V,+ V,) for synaptosomes from guinea-pig cerebral
cortex is 3.18fiIlmg of synaptosomal protein. V,, the matrix volume of the included
mitochondria, can be estimated from a volume of 0.8pI/mg of mitochondria1 protein
(Nicholls, 1978a) assuming that the included mitochondria contribute 10%to the total
synaptosomal protein (Nicholls, 1978a).
Eqn. (3) can only be solved for A i m if A y P is known. The synaptosomal plasma
membrane has a high electrical permeability for Rb+ (Blaustein & Goldring, 1975),
with the result that Rb+ should readily achieve a Nernst equilibrium across the plasma
membrane. However, in the absence of an ionophore such aivalinomycin, Rb+ will not
be accumulated by the included mitochondria. Thus :
Substitution for A y p in eqn. (3) therefore allows Aw, to be calculated.
In Table 1 this technique is applied to guinea-pig cerebral-cortical synaptosomes, and
the effects of additions that would be predicted to lower either A i mor A ypare examined.
In the control, the Rb+ distribution predicts a Atyp of 67mV, which is in good agreement
with published estimates for isolated synaptosomes (Blaustein & Goldring, 1975),
whereas the mitochondria maintain a A y , of 150V, similar to values obtained with the
isolated cortical mitochondria (Nicholls, 1978a). Addition of a low concentration of
proton translocator has littleeffect on hip,, but greatly lowers Ay,. In contrast, addition
of 60mM-KCI to the incubation medium decreases Atyp by 23mV, but has no effect on
Aw,. All synaptosomal preparations are to some extent contaminated with 'free'
mitochondria in addition to those within the synaptosome (Nicholls, 1978a; Booth &
Clark, 1978). However, the absence of substrate for the 'free' mitochondria, together
with the presence of 1.3 mM-CaZ+in the medium, should prevent a significant membrane
potential from being maintained across their inner membrane. Error due to accumulation of methyltriphenylphosphonium by these mitochondria should thus be negligible.
This work is supported by the Medical Research Council.
Blaustein, M. P. & Goldring, J. M. (1975) J. PhysioI. (London) 247, 589-615
Booth, R. F. G. & Clark, J. B. (1978) Biochem. J. 176,365-370
Lai, J. C. K., Walsh, J. M., Dennis, S. C. & Clark, J. B. (1977)J. Neurochem. 28,625-631
Nicholls, D. G. (1978~)Biochem. J . 170,511-522
Nicholls, D. G . (19786) Biochern. J . 176,463-474
Skulachev, V. P. (1970) FEBS Lett. 11, 301-308
1979