EMD - Calbiochem: Ionophores

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Ionophores

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Technical Overview I Figure 1: Mobile Ion Carrier I Figure 2: Pore-forming Ionophore I Products

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Ionophores promote the transfer of ions from an aqueous medium into a hydrophobic phase. They are small amphipathic molecules that dissolve in phospholipid bilayers and greatly increase their ionic permeability. The inner part of an ionophore is made of polar groups forming a tetra- or octahedral geometry that fits and encloses the desired ion. Around this polar cage are hydrophobic groups that allow the solubilization of the charged ion-ionophore complex in apolar solvents or lipid membranes. Ionophores shield the electric charge as the ion passes through the membrane, thereby providing a polar environment for the ion.

Some ionophores are natural products isolated from microorganisms while others are synthetic compounds tailored to a specific application. They are widely used in pharmaceuticals, in diagnostic radio-imaging, as plant and animal feed additives, and as chelators in biological and chemical research. Ionophores can be broadly classified as either mobile carriers (e.g., valinomycin), which diffuse forward and backward across the membrane, or as channel formers (e.g., Gramicidin) that form a tiny pore through the membrane. Both of these types operate by shielding the charge of the transported ion so that it can penetrate the hydrophobic interior of the lipid bilayer. Since ionophores are not coupled to energy sources, they permit net movement of ions only down their electrochemical gradients. The kinetics of ion transport by ionophores is rapid, with turnover rates of several thousand per second in biologic membranes. Charge makes a huge difference to the behavior of each carrier. The ionophore-ion complex, in some cases (e.g., valinomycin - K⁺), has a net electrical charge, whereas the unoccupied carrier is neutral. In other cases, only electrically neutral complexes are formed between the ionophore and the ion (e.g., Nigericin).

Valinomycin is an example of a mobile ion carrier. It is a ring-shaped polymer that transports K⁺ down its electrochemical gradient by picking up K⁺ on one side of the membrane, diffusing across the bilayer, and releasing K⁺ on the other side (Figure 1) In the absence of valinomycin (or any other K⁺ ionophore), K⁺ only rarely crosses a lipid bilayer. However, in the presence of valinomycin, K⁺ becomes freely permeable. A23187, a widely-used ionophore, is another example of a mobile ion carrier; however, it transports only divalent cations such as Ca²⁺ and Mg²⁺. It normally acts as an ionexchange shuttle, carrying two H+ out of the cell for every divalent cation carried in. When cells are exposed to A23187, Ca²⁺ enters the cytosol from the extracellular fluid down a steep electrochemical gradient.

Gramicidin is a good example of a channel-forming ionophore. Unlike mobile ion carrier ionophores, the pore forming ionophores are not sensitive to temperature changes. Gramicidin is a linear peptide of only 15 amino acid residues, all with hydrophobic side chains. It is the simplest and one of the best-characterized channel forming ionophore. It dimerizes in a head-to-head fashion to form a transmembrane channel that allows the electrogenic flux of monovalent cations, including protons. These dimers are unstable and are constantly forming and dissociating. The average open time for a channel is about 1 second. Hence, with a large electrochemical gradient, for each open channel gramicidin A can transport about 20,000 cations/millisecond, which is about 1000 times more ions than can be transported by a single mobile carrier molecule in the same time frame.

Characterization of the mechanisms involved in ion-transport by ionophores has played a crucial role in our understanding of the mitochondrial metabolism, proton gradient, and chemiosmotic coupling. The ionophores used to study mitochondrial metabolism generally belong to the mobile ion carrier group. Ionophores differ from uncouplers in that uncoupling agents increase the proton permeability and disconnect the electron transport chain from the formation of ATP, whereas ionophores act only to transport ions.

The distribution of ions between inner and outer phases in the presence of valinomycin has been a favorite method for measuring membrane potential, usually utilizing isotopically labelled Rb⁺. Under the influence of valinomycin, mitochondria will take up K⁺ at the expense of the proton gradient driven by coupled electron transfer or ATP hydrolysis (Figure 2). However, in the presence of a transportable anion (e.g., phosphate), or the anion of a permeable weak acid (e.g., acetate), the mitochondria will swell as they respond osmotically to the accumulation of salt. When an excess amount of K⁺ is present, the electrical component of the proton gradient disintegrates leading to severe biological effects. The carboxylic ionophores (e.g. monensin, salinomycin) have also been used in mitochondrial function studies. These ionophores mediate an electrically neutral exchange of cations for protons across cell membranes without employing ion channels. In the mitochondria, this discharges electrochemical gradients across the mitochondrial membrane, causing decreased ATP production and increased ATP utilization, leading to cell death.

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Ionophores

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