Michael J. Selwyn

Calcium transport in mitochondria

Since Vasington and Murphy [ 1, 2] and DeLuca and Engstrom [3] found that respiriglg mitochondria accumulated massive amounts of Ca 2+ ions a great deal of work has been done on the energy-linked accumulation of divalent cations by mitochondria, (Lehninger, Carafoli and Rossi [4]). It has been shown that when energy is available, either from electron transport or from ATP hydrolysis, mitochondria can accumulate not only Ca 2+ but also Sr 2+, Mn 2+ and, to a lesser extent Ba 2+ ions against a very high concentration gradient. This has been interpreted [5, 6] to mean that the mitochondrial membrane contains a Ca 2+ pump or CaZ+/2H + exchange pump which is driven by the non-phosphorylated high energy intermediate. An alternative explanation proposed by Mitchell [7] is that the membrane contains a Ca 2+ carrier or Ca2+/H + exchange diffusion carrier, and that Ca 2+ ions are accumulated in response to the pH and/or electrical potential difference across the membrane. It has also been reported that mitochondria are able to bind Ca z+ in the absence of an energy source but the relation between this binding and the energylinked massive accumulation is not clear, nor is it clear whether the binding is on the surface of the membrane or inside the mitochondria [8]. The existence of carriers for monovalent cations and for a variety of anions has been investigated by suspending mitochondria in iso-osmotic solutions of salts [9], for when both anion and cation are permeable the solution is osmotically inactive and the mitochondria swell. Furthermore the nature of the translocation of the~two ions must be complenmntary such that no electrical charge or pH imbalance is produced by passage of the ions into the mitochondria. Mitchell and Moyle. [ 10] have established that chloride crosses the mitochondrial membrane slowly by electrogenic uniport and that thiocyanate does so rapidly. Acetate crosses the membrane either as acetic acid or on an acetate/hydroxide anti-porter, the net effect being the same in either case, i.e. :an electrically neutral process which produces a ~ a i f f~rea~ across the membrane. Several non-penetrant anions have been reported but none appears very satisfactory for investigations with divalent metal ions. Investigation showed that rat liver,mitochondria have a very low permeability to isethionate~an anion which is present in high concentrations in squid axons and therefore likely to be physiologically inert. Furthermore most salts of this ion are very soluble. The set of anions, isethionate, acetate, chloride and thiocyanate, thus provides a means for investigating penetration properties of cations and in particular for testing the validity of Mitchells suggestion about the divalent cation carriers and, if they are driven by electrochemical gradients, for investigating the nature of the carrier.

Dependence on Na+ of control of cytoplasmic pH in a facultative alkalophile

Regulation of the cytoplasmic pH of Exiguobacterium aurantiacum is dependent on the presence of Na+ in the medium. The data suggest that above 500 μM external Na+ the cells are able to regulate the cytoplasmic pH by the operation of a Na+ cycle involving a Na+/H+ antiport and a route for rapid Na+ entry. Our data indicate that the rate of entry of Na+ is subject to control by the cytoplasmic pH via feedback inhibition.

Palmitoyl‐CoA inhibits the mitochondrial inner membrane anion‐conducting channel

Palmitoyl‐CoA is shown here to inhibit the pH‐dependent anion‐conducting channel (IMAC) in the inner membrane of rat liver mitochondria, with half‐maximal inhibition at 2.4 μM. It has little effect on the transport of ribose, thiocyanate and glutamate. Palmitic acid and palmitoyl‐carnitine stimulate the entry of all the above metabolises. CoASH and carnitine have no effect on chloride uniport. Palmitoyl‐CoA and the IMAC may have a role in controlling thermogenesis in liver mitochrondria.

Effect of some organometallic compounds on the permeability of chloroplast membranes.

Previous work in this laboratory has shown that trialkyltin compounds mediate an anion-hydroxide exchange across mitochondrial, erythrocyte and liposome (smectic mesophase) membranes [ 1,2] . Kahn [3] has reported that trialkyltin compounds inhibit and uncouple photophosphorylation in isolated chloroplasts. Siegenthaler [4] and Murakami and Packer [.5] have reported that when phenyhnercuric acetate is added to chloroplasts in a chloride medium, the light-induced light scattering and volume changes resemble those in an acetate medium [6]. Scott et al. [7] have found that organomercury compounds alter the permeability properties of the mitochondrial membrane. These reports prompted investigation of the effect of organometallic compounds on the permeability properties of the chloroplast membrane, and an extension of our studies on liposomes to the effects of organomercury compounds. Our observations show that tripropyltin chloride and phenylmercuric acetate mediate a chloride-hydroxide exchange across the thylakoid membrane.

Inhibition of electrogenic anion entry into rat liver mitochondria by N,N'-dicyclohexylcarbodiimide

The carboxyl group reagent dicyclohexylcarbodiimide inhibits the electrogenic entry of Cl− and NO3 − into rat liver mitochondria at alkaline pH. The inhibition is time dependent and 50% inhibition is obtained by the addition of 3–4 nmol DCCD/mg protein. The blockage of the pH‐dependent anion‐conducting pore appears to be unrelated to the other known actions of DCCD on rat liver mitochondria but seems similar to its effect on the uncoupling protein of brown adipose tissue.

Inhibition of mitochondrial anion permeability by local anaesthetics

Local anaesthetics such as nupercaine have been shown to have a protective action on the structure and energy-linked functions of rat liver mitochondria during incubation at 30°C or prolonged storage at 0°C [ 1.21. This action has been attributed to the inhibition of endogenous Ca’+-activated phospholipase A by the local anaesthetics [3,4], These observations have provided a logical basis for the incorporation of nupercaine in the medium for experiments on mitochondrial Ca*+ uptake [5] in order to overcome the problem of mitochondrial fragility encountered in such experiments 161. Local anaesthetics appear to have other actions on mitochondria such as competition for cation binding sites [7] and stimulation of Ca*+ uptake [8,9]. Although rat liver mitochondria are usually considered to have a low permeability to most anions, other than by highly specific carriers [IO] , rat liver mitochondria have been reported [ 1 l] to become permeable to Clat high pH (pH 8-9) and conditions reported [ 121 under which beef heart mitochondria become permeable to Br-, succinate, fumarate and citrate as well as Cl-. We have reported [ 131 investigations of the anion permeability of rat

Role of Na+ in pH homeostasis by the alkalophilic bacterium Exiguobacterium aurantiacum

Summary: Exiguobacterium aurantiacum, a facultative alkalophile, can maintain a δpH of up to 1·7 pH units, acid inside, and can rapidly adjust the cytoplasmic pH (pHi) in response to a shift in external pH (pHo), demonstrating effective pHi homeostasis. The presence of Na+ accelerated the attainment of a new steady-state pHi during a shift in the alkaline direction but slowed the attainment of new steady state following a shift in pHo in the acid direction. Measurements of internal Na+ following the addition of 6 mm-NaCl to cells incubated under conditions whereby the cells either could (+ 0·68 mm-NaCl) or could not (0·08 mm-NaCl) regulate pHi indicated that pHi exerted some feedback control over Na+ influx. A model for the involvement of Na+ in pHi regulation comprising an electrogenic Na+/H+ antiporter and a sodium influx channel regulated by pHi is proposed. Intrinsic to this model is the suggestion that the Na+/H+ antiporter is not the sole site of feedback control by pHi.

Decrease of proton permeability of CF1-deficient chloroplast particles by triphenyltin

Removal of the mitochondrial ATPase (F, ) has been reported to increase the proton permeability of sub-mitochondrial particles [ 1,2] . Mitchell [3,4] suggested that removal of Fr unblocked a natural proton conducting channel through the F. component of the coupling membrane. Oligomycin, an inhibitor of the membrane-bound mitochondrial ATPase has been found to decrease the high proton conductivity of Fr -deficient sub-mitochondrial particles and bacterial chromatophores [ 1,4,5,6,7,8,9] . Similar studies on isolated chloroplasts and bacteria [lO,l I] have shown that removal or absence of the coupling factors rendered these particles permeable to protons and that titration with Nfl-dicyclohexylcarbodiimide restored them to a ‘pseudo-coupled’ state. Unlike oligomycin, triorganotin compounds have been found to inhibit photosynthetic as well as oxidative phosphorylation reactions [12,13,14,15,16,17,18]. Kahn [ 151 showed that chloroplasts which had been uncoupled by treatment with EDTA had a low residual level of light-induced proton uptake which was increased by the addition of tributyltin and he also found that the slow residual rate of photophosphorylation in EDTA-uncoupled chloroplasts was enhanced by tributyltin. However, Kahn did not show whether blockage of proton leakage or restoration of proton pumping was responsible for the increased

Respiration driven Cl− uptake by submitochondrial particles

Both Mg2+ and oligomycin are required for the establishment of a membrane potential and the uptake of Cl− in submitochondrial particles prepared from rat liver. The effect of oligomycin is considered to be due to blocking of H+ conduction through exposed F0 channels of the ATPase complex whereas Mg2+ may more directly affect the anion‐conducting channel.