The Ions

1.1 INTRODUCTION

Only sodium (Na), potassium (K), magnesium (Mg) and calcium (Ca) among the Group 1 and 2 elements are essential in biological systems. Some of the other s-block elements are used in medicine (e.g. lithium, Li and barium, Ba) and/or occur as minor (but useful) contaminants in calcium biominerals (e.g. strontium, Sr).

The metal ions with which we are involved in this book (Na+, K+, Mg2+ and Ca2+) are less striking than many of the other metal ions that have significant biological roles. Metal ions such as iron and copper are coloured in solution, have more than one common oxidation state and are strongly coordinating, all properties lacking in the s-block metals. Nevertheless, the very lack of these properties enable them to be the workhorses in many biological processes. These metals display only one stable oxidation state resulting from the loss of one (M+, Group 1) or two (M2+, Group 2) s-electrons. This enables the metal ions to move around the cell without any danger of being oxidized or reduced. In this way they play many vital, complex and intriguing roles. They aid in neurologic and neuromuscular conduction, help regulate pH, maintain osmolality of body fluids, are involved in muscle contraction and are required for the functioning of many enzymes. They are components, calcium in particular, of many of the solid structures in most organisms.

The Heart Says It All

Animal tissues can be kept alive for experimentation for a short time by immersion in a buffered solution that mimics the ionic composition of animal plasma (Ringer's solution). A graphic illustration of the importance of these ions is shown by the composition of a cardioplegic solution used to preserve a donor heart (for 4-6 hours) prior to a transplant. The solution typically contains NaCl, 144 mM; KCl, 20 mM; MgCl2, 16 mM; CaCl2, 2.4 mM and procaine (a local anaesthetic that blocks nerve sodium channels, see 4.6), 1.0 mM at a pH of 5.5-7.5 at 4 °C.

1.2 OCCURRENCE

1.2.1 Earth's Crust

The s-group metals Na, K, Mg and Ca are, after Al and Fe, the most abundant metals in the earth's crust.

An excess of sodium salts in the soil can cause severe problems in crop production. This is because an influx of sodium ions into plant cells can upset critical biochemical processes by competing with potassium ions in membrane transport and enzyme activation, and can also cause osmotic stress. It has been estimated that about one-third of the world's irrigated land is unsuitable for growing crops because of high sodium ion contamination. Calcium ions, on the other hand, can be beneficial in that they help to maintain or enhance the selective absorption of K ions by plants.

A Possible Solution to Salt Contamination of Soil

Shrubs of the genus Plantago produce seeds that are used as a food for birds and also as a bulk-forming laxative. Plants, even those of the same species, have differing abilities to tolerate salty soil. Plantago mahtima, but not P. media, is salt tolerant. P. mahtima (but not P. media) contains an Na+/H+ antiport protein that permits the interchange of Na+ and H+ions (3.6.1). This allows the plant to sequester Na+ ions in the large intracellular vacuole and away from the cytosol where salt interferes with metabolic processes. This represents one way of defeating a saline environment.

A salt-tolerant Arabidopsis has been engineered by overexpressing a single endogenous gene, AtNHXl, which encodes for an Na+/H+ antiport protein. Similarly, tomato plants have been genetically engineered to produce high levels of an Na+/H+ antiport. These plants flourish in 200 mM salt water, and produce good-looking and tasty fruit that is low in sodium even though the leaves accumulate large amounts of the ion. It could now be possible to produce a whole array of salt-tolerant crop plants, which would enable the use of seawater for irrigation.

1.2.2 The Seas

Most (about two-thirds) of the earth's surface is covered with water, and in the seas the s-group metal ions specified above are the major cations present. On average, there is 3.5 g of salts per 100 mL of seawater. This rises to 4% in the Mediterranean and off the southwest coast of Crete, where near-dry conditions 3-5 million years ago has produced a brine pool with a very high MgCl2 concentration. The great salt lake in Utah is saturated with salts and NaCl crystallizes on the shore. In the Dead Sea, CaSO4 crystallizes out. Nevertheless, certain creatures, tiny crustacean brine shrimp (Artemia) for example, but not fish, can survive in these conditions. Indeed, Artemia cannot live for long in fresh water. Coastal evaporation ponds are a commercial source of Artemia, which is used as fish food.

1.2.3 Biological Materials

The s-group metal ions Na, K, Mg and Ca are found in most cells in mM concentrations, see Table 1.1. It is often very difficult to measure the intracellular concentrations, particularly of the free ions. Inorganic and organic anions, which are lower in concentration outside the cell than inside, maintain cell neutrality.

One can draw one's own conclusions about the significance, if any, of the similarity between the concentrations of these ions in extracellular media and in seawater in which life may have evolved. Although there are significant differences between ion concentrations in vertebrates and invertebrates, the differences between the solute composition of the cells and of the extracellular environment, with the glaring exception of Mg, persist for squid and for mammals. These concentration differences are used to carry out many of the vital tasks that are necessary to maintain life.

1.3 COORDINATION CHEMISTRY

1.3.1 Ion Sizes

There is some disagreement as to the sizes of the ions of the s-block (particularly of Li+). However, Pauling's values have, by and large, stood the test of time and are shown in Table 1.2.

We will find that ion size is particularly relevant when the transfer of ions through channels is being considered. For example, a long-standing vexing question arises as to how a dehydrated Na+ ion, which is substantially smaller than a K+ ion, can be rejected by the very selective K+ channel. Earlier proposals have now been strongly supported and extended by an X-ray crystallographic examination of the K+ channel (KcsA) cloned in mg amounts from the membrane of the bacterium Streptomyces lividans. For more about the channel structure and passage of K+ through the pore see 3.4.2. The constriction (~ 3Å across) at the selectivity filter demands that all coordinated H2Os are stripped from the hydrated ion for passage of the ion through the channel. An ideal fit and coordination by the channel O atoms compensate for the associated energy loss. These criteria apply to K+ ions much better than to Na ions. This accounts for an ~102 preference for K+ ion entry (see Figure 1.1).

1.3.2 Donor Atoms and Strength of Binding

The s-block cations are highly electropositive and in the formation of complexes with smaller ligands have a preference for those with O-donor atoms. This generalization applies equally well to their coordination in metallobiomolecules where the ligands are polypeptides, proteins, nucleotides (1) and polynucleotides. In these ligands the O atoms are provided by the RCO-2 and OH groups from the side chains of amino acids, -C = O from peptide bonds and PO3-4, HPO2-4, H2O or OH- moieties. Some illustrative formation constants and comments are shown in Table 1.3 (see Structure (1)).

Many enzymes require the presence of a +1 cation, particularly K+ (2.2.2), for activity. The interactions of Na+ and K+ with these enzymes is unlikely to be strong and therefore the metal ion is not an integral part of the enzyme. In the several crystal structures that have been solved, the first coordination sphere around Na+ or K+ has been occupied predominantly by oxygen atoms.

The atoms that coordinate to Mg2+ and Ca2+ are a much more important consideration as these ions form much stronger complexes, see Table 1.3. A startling illustration of the stronger binding power of Ca+ over Na+ is in the behaviour of mucus. Mucus is a slimy material rich in the glycoprotein mucin. It is an effective lubricant in the mouth, stomach, etc. and aids in the transfer of dirt and bacteria from the lungs to the mouth. It is stored as compact granules in the secretory vesicles within goblet shaped cells. The condensed polymer gel is maintained by high Ca+ (and H+) ion concentrations. These ions bind to the negative charges on the sulfate and sialic termini (acidic sugars found in many glycoproteins) of the long mucus strands. Mucus is released by intracellular calcium ([Ca2+]i) regulated exocytosis (5.1). The tightly packed mucin rapidly springs open as the Ca2+ ion concentration falls and is replaced by Naions. The sodium ions cannot hold the mucin together, so due to negative charge repulsions, mucin goes to the expanded state in an explosive release. The mucin network in the giant granules of the slug expands approximately 600-fold in 20-30 ms!

Suffocating Mucus is Used as a Weapon by Hagfish

The hagfish is an eel-like scavenger that can attack and suffocate predator fish using mucus. Hagfish have about 150 slime glands along their sides. When provoked the gland contents are ejected rapidly into the surrounding sea. The contents include the mucin-packed vesicles that break open. The ejected slime (about 5 g), which is a mixture of mucus (expanded mucin) and thin strong fibres that act as a scaffold, is an effective trap. Hagfish can strip fish (live, dead or dying!) so entangled using a rasp-like tongue.

Oxygen donor atoms are strongly preferred by Mg ions and these emanate from phosphate groups in DNA, RNA, ribozymes, etc. Sometimes this coordination is reinforced by attachment of a nucleotide base N atom. This interaction is probably the most important impact made by Mg ions in biology.

Diagnoses of Illnesses Could be Simplified

In the polymerase chain reaction (PCR) a target DNA segment with a genetic sequence specific for a particular organism can be amplified millions-fold using a specific primer (oligonucleotide), DNA polymerase and the four deoxyribonucleoside triphosphates.

If the blood of a patient contains the target DNA arising, for example, from a bacterium, then by applying PCR, conversion of the nucleotides into DNA will occur and the bacterial-induced illness can be diagnosed. A method suggested for the detection of the PCR-amplified material uses the release of Mg2+ ions (and the attendant conductivity increase) that accompanies the conversion of the nucleotides to DNA. The nucleotides, with three phosphate groups, bind more strongly to Mg2+ ion than does DNA, with only one phosphate group per base.

The binding of four N atoms from the corrin ring in chlorophyll to Mg+ a rare example of nitrogen coordination in a biomolecule with an s-block cation.

1.3.3 Calcium Binding Domains

Only O-donor atoms are known so far for Ca2+ ion binding to biomole-cules. Two Ca+ ion-binding motifs in particular are favoured in hundreds of proteins with widely varying functions. These are the EF hand, typified by the calcium site in calmodulin, and C-domains. The binding of the latter to phospholipids greatly enhances the strength of Ca+ ion binding (see below).

There are two globe-shaped regions connected by a seven-turn α-helix at the N- and C-termini of calmodulin. Each globular region contains two Ca2+ ion binding sites. The amino acid sequence at each Ca+ ion binding site is highly conserved in the proteins from various animals and plants. Each calcium ion is seven-coordinated, with the O atoms provided by side chain carboxylate (glu, asp), carbamoyl (gin, asn) and alcohol or peptide C=O (thr, ser, tyr) groups and H2O. These originate from the 12 residue-containing loop in the E helix-loop-F helix motif (also called the E-F hand), that is also found in other calcium binding proteins (2.5). The E helix-loop-F helix motif contains a sequence of amino acids that is a useful signature for locating similar new proteins. This was one of the first structural motifs to be recognized in protein structures.

The other important Ca2+ binding entity is a so-called C2-domain. This domain also binds to phospholipid and this combination enhances Caion binding by 103-fold. In turn, Ca2+ ions promote membrane binding by C2-domains. The C2-domain, about 130 residues in length, is present in a wide range of proteins, e.g. protein kinase C, cytosolic phospholipase A2, PLCδ1 (3.5.2) and synaptotagmin (2.5.3). In the C2-domain, multiple Caions bind in clusters. The residues involved are often aspartate side chains acting as bidentate ligands. PLCδ1 contains both E-F-hand and C2 domains as well as catalytic sites.

1.3.4 Geometry of Binding

The regular geometries encountered with small complexes of these metals (octahedral coordination is most favoured) are not generally duplicated in metalloproteins because of the constraints imposed by the protein structure. Sometimes the s-block metal ion may even be compelled to bind to an N-donor, which has been forced into a coordination position by the protein.

Dialkylglycine decarboxylase (DGD) is a pyridoxyl phosphate-dependent enzyme that catalyses decarboxylation and transamination in a catalytic cycle (Equation 1).

(CH3)2C(NH2)COOH + CH3COCOOH [right arrow]

CH3CH(NH2)COOH + CO2 + (CH3)CO (1)

Crystal structures of the enzyme with either a K+ or an Na+ ion in position near the active site have been very informative about the geometries and the roles of the univalent metal ions in the catalysis. In DGD, six oxygen atoms are octahedrally disposed around the K+ ion (M-ligand ~ 2.73 Å) whereas only five oxygens form a distorted trigonal bipyramid around the Na+ ion (M-ligand ~ 2.33 Å), Figure 1.2.

The change of geometry around the Na+ ion induces marked changes in the orientation of tyr-301, which probably interacts with bound substrates, and in ser-80, which is a K+ but not an Na+ ligand. These changes disrupt the nearby active site and can be used to rationalize the facts that Na+ ion is an inhibitor and K+ ion an activator of the enzyme.

The ionic radius of Mg22+ is much smaller than that of Ca2+ (Table 1.2). Usually the protein is not flexible enough to adjust the coordinating O atoms to the smaller Mg2+ ion. As a consequence, fewer O atoms from the protein are likely to coordinate to the Mg2+ ion (preferred coordination number of six) without strain, compared to the Ca2+ ion (coordination numbers of 6–8 common). This is the reason that the Ca2+ ion forms the stronger, more diverse complexes. The differences in the geometries exhibited by Ca2+ compared to those of the Mg2+ ion may cause different structural perturbations when each of these ions is associated with an enzyme. This idea has been used to rationalize the 10-3 difference in rates when an Mg2+ ion is replaced by Ca2+ ion in the reaction catalysed by isocitrate dehydrogenase in which Mg2þ is the endogenous ion in vivo (Equation 2).

1.3.5 Kinetic Lability

The hydrated Na+, K+ and Ca2+ cations are extremely labile – that is their coordinated water molecules exchange with solvent water at rates in excess of 108 s-1. The replacement of coordinated water by ligands is therefore facile and unlikely to be a rate-limiting step. This is particularly important if the triggering action of Ca2+ ions is to be effective. In contrast the water exchange rates for Mg2+ ions are much slower (~105 s-1).

1.4 SOLUBILITY OF SALTS AND ATTENDANT PROBLEMS

Most common sodium and potassium salts are soluble in water, although the solubilities cover a wide range. Therefore the presence of N+ or K+ ions even in high concentrations in the cellular milieu, which contains several anions, usually causes no precipitation problems. An exception to this is in hyperuricemia where the offending anion is urate (2) (2,6,8-trioxypurine anion).

A Pain in the Big Toe

In higher primates the final product of purine degradation is uric acid, which is excreted in the urine. The overproduction, or impaired elimination, of uric acid leads to elevated amounts in serum (hyperuricemia) and urine. This results in the deposition of sodium urate crystals in extreme joints, e.g. the big toe, which is the inherited disease gout. A mixture of uric acid and sodium urate (kidney stones) can promote kidney disease. These problems only occur in humans, apes and the Dalmatian dog (!) because they lack or are unable to use the enzyme uricase, which in other mammals catalyses the further degradation of uric acid to very soluble allantoin (3).