Alan P. Dawson

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.

Inositol (1,4,5)trisphosphate-promoted Ca2+ release from microsomal fractions of rat liver.

Crude mitochondrial fractions containing a substantial amount of microsomes accumulate Ca2+ in the presence of ATP, ruthenium red and oligomycin. A proportion of this accumulated Ca2+ is released by the addition of low concentrations (ca. 1 microM) of inositol (1,4,5) trisphosphate . Under some conditions the release is transient, and evidence is presented which suggests that this is due to inhomogeneity in the vesicle population. (1,4,5)inositol trisphosphate -induced Ca2+ release can also be demonstrated, under appropriate experimental conditions, in a more purified microsomal fraction essentially free of mitochondria.

GTP enhances inositol trisphosphate-stimulated Ca2+ release from rat liver microsomes

Low concentrations of GTP (10–50 μM) greatly enhance the inositol 1,4,5‐trisphosphate stimulated Ca2+ release from rat liver microsomal vesicles. The effect of GTP depends on the presence of low concentrations of polyethylene glycol in the incubation medium. Guanylyl imidodiphosphate is ineffective at mimicking the GTP effect and inhibits the action of GTP added subsequently.

Heparin inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from rat liver microsomes

Inositol 1,4,5‐trisphosphate (Ins (1,4,5)P3)‐stimulated Ca2+ release is inhibited by low concentrations of heparin (IC50=4.5 μg/ml). GTP‐stimulated Ca2+ release is unaffected at a heparin concentration of 16 μ/ml. Addition of heparin after Ins (1,4,5)P3 causes the rapid re‐uptake of Ins (1,4,5)P3‐releasable Ca2+.

Modulation of type-1 Ins(1,4,5)P3 receptor channels by the FK506-binding protein, FKBP12.

FK506-binding protein (FKBP12) is highly expressed in neuronal tissue, where it is proposed to localize calcineurin to intracellular calcium-release channels, ryanodine receptors and Ins(1,4,5)P(3) receptors (InsP(3)Rs). The effects of FKBP12 on ryanodine receptors have been well characterized but the nature and function of binding of FKBP12 to InsP(3)R is more controversial, with evidence for and against a tight interaction between these two proteins. To investigate this, we incorporated purified type-1 InsP(3)R from rat cerebellum into planar lipid bilayers to monitor the effects of exogenous recombinant FKBP12 on single-channel activity, using K(+) as the current carrier. Here we report for the first time that FKBP12 causes a substantial change in single-channel properties of the type-1 InsP(3)R, specifically to increase the amount of time the channel spends in a fully open state. In the presence of ATP, FKBP12 can also induce co-ordinated gating with neighbouring receptors. The effects of FKBP12 were reversed by FK506. We also present data showing that rapamycin, at sub-optimal concentrations of Ins(2,4,5)P(3), decreases the rate of calcium release from cerebellar microsomes. These results provide evidence for a direct functional interaction between FKBP12 and the type-1 InsP(3)R.

Calcium signalling: How do IP3 receptors work?

The intracellular receptor for inositol 1,4,5-trisphosphate (IP3) is responsible for generation and control of very complex Ca2+ signals. New experimental approaches to studying the kinetics of the IP3 receptor are now beginning to give some insight into the mechanisms behind its rather bizarre properties.

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels

Ca2+ within intracellular stores (luminal Ca2+) is believed to play a role in regulating Ca2+ release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-gated Ca2+ channel (or Ins(1,4,5)P3 receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P3 receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca2+] using K+ as the current carrier. At a high luminal [Ca2+] and in the presence of optimal [Ins(1,4,5)P3] and cytosolic [Ca2+], a short burst of Ins(1,4,5)P3 receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca2+] caused the channel to reactivate indefinitely. At luminal [Ca2+], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca2+] caused reactivation. We provide evidence that luminal Ca2+ is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca2+ overload is prevented when the energy state of the cell is compromised.

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.

Regulation of Intracellular Ca2

Publisher Summary Intracellular Ca 2+ plays an important role in controlling processes such as muscular contraction and secretion. The study of the mechanisms regulating Ca 2+ concentrations in cells has evolved in parallel with studies on the processes regulated by Ca 2+ . The discovery of the relationship among receptor-activated polyphosphoinositide breakdown, inositol 1,4,5-trisphosphate, and mobilization of intracellular Ca 2+ has given great impetus to this area of investigation and has encouraged the study of a wide variety of cell types. The regulation of intracellular Ca 2+ concentration requires interactions between a variety of transport systems in several different membranes. The chapter discusses the Null-point technique for the determination of intracellular Ca 2+ concentration. The technique involves the suspension of hepatocytes in media containing various concentrations of free Ca 2+ and a metallochromic Ca 2+ indicator, Arsenazo III to permeabilize the plasma membranes of the cells with digitonin and to measure whether Ca 2+ has been accumulated or released from intracellular stores.

Specificity of the purified inositol (1,3,4,5) tetrakisphosphate-binding protein from porcine platelets

The specificity of the inositol 1,3,4,5‐tetrakisphosphate binding protein purified from porcine platelets [Cullen et al. (1995) Biochem. J. 305, 139–143] was examined using all the isomers of myo‐inositol tetrakisphosphate. From the relative potencies of these compounds it appears that phosphorylation of the 1, 3 and 5 positions is essential for high affinity binding, that there is some tolerance of phosphorylation of the 6‐hydroxyl, but none of a phosphate in the 2‐position, and that phosphorylation of the 4‐hydroxyl has very little influence. The binding of Ins(1,3,4,5)P4 was not appreciably altered by physiological Mg2+ concentrations, and the pH dependence of binding under physiological conditions showed a decline from pH 5.5 to pH 9.0.