Evidence for operation of the direct zinc ligand exchange mechanism for trafficking, transport, and reactivity of zinc in mammalian cells

Abstract

In addition to its critical role in normal cell function, growth, and metabolism, zinc is implicated as a major factor in the development and progression of many pathological conditions and diseases. Despite this importance of zinc, many important factors, processes, and mechanisms of the physiology, biochemistry, and molecular biology of zinc remain unknown. Especially important is the unresolved issue regarding the mechanism and process of the trafficking, transport, and reactivity of zinc in cells; especially in mammalian cells. This presentation focuses on the concept that, due to the existence of a negligible pool of free Zn(2+) ions in the mammalian cell environment, the trafficking, transport and reactivity of zinc occurs via a direct exchange of zinc from donor Zn-ligands to acceptor ligands. This Zn exchange process occurs without the requirement for production of free Zn(2+) ions. The direct evidence from mammalian cell studies is presented in support of the operation of the direct Zn-ligand exchange mechanism. The paper also provides important information and conditions that should be considered and employed in the conduct of studies regarding the role and effects of zinc in biological/biomedical research; and in its clinical interpretation and application.

Figure 1

The kinetic properties of Zn-Ligands in Zn-uptake transport by prostate PC-3 cells (modified from [11]). A. Zinc uptake as a function of ZnCl₂ (i.e. free Zn²⁺ ion) concentration. The Km value was derived from Lineweaver-Burk application. B. Comparison of Zn uptake from free Zn²⁺ ion concentration vs., total Zn as Zn-Ligands. C. Effects of titration of ZnCl₂ with increasing concentrations of citrate on the concentration of free Zn²⁺ ions and the Zn uptake transport. D. Comparison of Zn65 uptake and C14-citrate uptake from Zn-Citrate.

Figure 2

Representation of the direct Zn ligand transport process as applied to: A. plasma membrane zinc uptake transporters (e.g. ZIP-family transporters); and B. intracellular organelle transporters (e.g., ZnT-family transporters). The representation shows Zn exchange directly between Zn-Ligands and Zn-transporter, in the absence of formation of free Zn²⁺ ions.

Figure 3

Transmembrane domains 3, 4 and 5 and the large loop of hZip1 are represented. The histidines (H) in the loop are conserved in ZIP family transporters. Mutation of histidines 158 and 160 significantly decreased zinc transport. The histidines in the loop and histidines 190 and 217 in transmembrane domains 4 and 5, respectively potentially form a zinc coordination-binding site that functions in the transport process.

Figure 4

Comparison of Zn uptake from Zn-Ligands and from free Zn²⁺ ions in PC-3/ZIP1 cells vs. PC-3/mut ZIP1 cells. Zinc uptake is decreased in ZIP1-mutant cells compared to PC-3 ZIP1 wild-type cells. In both cases, Zn transport is dependent upon total Zn concentration and not on free Zn²⁺ ion conc. Mutations were the histidines in the TM3-4 loop as shown in figure 3.

Figure 5

Kinetic studies of zinc uptake transport by prostate mitochondria. A. Time-dependent uptake of Zn from Zn-Ligands as a function of total Zn vs. free Zn²⁺ ion. B. Concentration-dependent zinc uptake from Zn-Ligands vs. from free Zn²⁺ ion; and corresponding Km values. C. Effect of titration of ZnCl₂ source of free Zn²⁺ ions by increasing concentrations of citrate on Zn uptake as a function of total Zn concentration vs. free Zn²⁺ ion concentration. D. Comparison of Zn65 uptake and C14-citrate uptake from Zn-Citrate. E. Effect of Ca²⁺, Mg²⁺, and Cd²⁺ on Zn transport.

Figure 6

Zinc uptake studies with liver mitochondria. A. Comparison of Zn uptake from Zn-Ligands vs. free Zn²⁺ions. B. Comparison of Zn accumulation by liver and prostate mitochondria. C. Comparison of Zn uptake by in-tact mitochondria and mitoplasts. D. Comparison of Zn accumulation from Zn-Metallothionein and Zn-Cysteine by liver and prostate mitochondria.

Figure 7

Comparative effects of free Zn²⁺ ions and Zn-Ligands on the respiration of liver and prostate mitochondria. A and B. Tracings obtained following five minute incubation with Zn solution to the mitochondrial preparation. Numbers at tracings are relative respiration rates. ZnCl, ZnCit, ZnCys have similar rates. C. Tracing begun prior to succinate and Zn treatments to obtain immediate effects Zn on respiration.

Figure 8

The effect of Zn from Zn-Ligands on terminal oxidation of liver mitochondria. A. Zn effects on succinate oxidation via Complexes II and III. B. Zn effects on cytochrome oxidase (Complex IV) activity.

Figure 9

Representation of the direct Zn intermolecular exchange process. The representation eliminates the involvement or requirement for the production of free Zn ions for the Zn transfer mechanism. Although this representation has been applied to protein—protein interaction, we propose that it also applies to LWM Zn-Ligand--protein exchange of Zn.

Figure 10

Titration of 100 μM apo-CP (CCHH) with different concentrations of rabbit Zn₇MT-2. pH 7.4. Results show that only one Zn site donates Zn from Zn₇MT to the acceptor protein. (Taken from [31] with permission from Elsevier Science)

Figure 11

Illustration of the transport and trafficking of Zn from blood plasma to mitochondria by the direct Zn-Ligand exchange mechanism. LMW donor Zn-Ligands and Zn-MT directly transfer Zn to the Zn uptake transporter proteins transporters and to other Zn acceptor zinc ligands. No formation of free Zn²⁺ ions is involved.