Examples from Nature

Naturally occurring transport systems are the most interesting ones because the majority of them are actually proteins. There are also minimalistic channels and carriers.

The term “natural ion channel” is somehow bound with the work of the MacKinnon group.^3,15–17 This group has extensively studied K⁺ channels by the means of crystallography. Several discoveries in this field come from this group. Arguably the most important contribution concerns the identification of the “channel” binding site in the middle of the protein bundle (Figure 1).

These perfectly aligned peptides allow the mimicking of the water coordina-tion. In fact, the protein mimics the potassium coordination with water and, as a consequence, sodium could enter but would collapse the structure. This selectivity creates a perfect K⁺ channel that filters other cations. Figure 1 depicts two crystal structures that belong to two different K⁺ channels, but there is no need to specify these K⁺channels as the “K⁺filter” is a common structure found ubiquitously.

In addition, the K⁺at the bottom of the filter is not held by a coordination to any carbonyl group, because at this stage K⁺is held in place and directed by the cooperative action ofα-helix bundles that “coordinate” the K⁺cation with cation-macrodipole interactions.

This short description should give an idea of the complexity of protein trans-port systems. Voltage gating was not addressed in the discussion but has also been explored.¹⁷Selectivity as well as stimulus-responsiveness are achieved by conformational changes of thewhole protein with the corresponding changes in the “filter” region.

Another example for natural “channels” can be found with the anthrax toxin. Actually, in order to fulfill its function, in this case to kill the cell, the toxin needs to deliver either the lethal factor (LF, 90 kDa) or/and the edema

Figure 1:Crystal structure of the KvAP K⁺channel (axial view)¹⁷(left) and zoom-in zoom-into the crystal structure of the KcsA K⁺channel with K⁺coordination (side view)¹⁶(right).

factor (EF, 89 kDa) into the cytoplasm. This is achieved by the protective antigen (PA, 83 kDa) which forms a heptameric structure as shown in Figure 2.

This heptameric structure is aβ-barrel pore with a pore size of 20 - 35 ˚A. At this point, the LF can traverse the lipid membrane. The action of the anthrax toxin does not end here and other steps are needed. In addition, an alternative path that involves endocytosis of the PA (modified) bind with both LF and EF followed by heptamerization in the late endosome and delivery of both factors into the lumen.^18,19

This example of a pore should illustrate the degrees of complexity that gov-erns natural systems as well as illustrate, in the extreme case, the differences between channels and pores.

Of course, not all transport systems that nature uses are based in proteins:

simple peptides can also promote transport. Given the context, the description of another toxin is fitted: melittin.

Melittin is one of the components of bee toxin. It is a relative simple pep-tide with for sequence Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH₂and, aside from the physiological effects that make it a very toxic molecule, it is able to form

1 Introduction 5

Figure 2:Crystal structure of the (PA63)7 prepore structure monomer (left) and heptamer of the (PA63)7prepore structure from the bottom (right).¹⁸

channels when interacting with the lipid bilayer membrane.²⁰

Melittin form channels (or pores) in the lipid bilayer membrane as shown by the characteristic signature in planar membrane conductance experiments, but these are not unimolecular but rather the association of around eight α-helix units as shown by several spectroscopic methods like circular dichroism.²¹ In addition, the aggregation of melittin units does disrupt the membrane, which, at high concentrations, leads to micellar aggregates and therefore its channel formation mechanism is said to be “micellar” or “toroidal”.^7,20

The arrangement of the melittin units is parallel between each other, and parallel to the phospholipids. The presence of positive charges near to the C-terminus make it more selective towards anions than cations.

Similar to melittin is the also naturalalamethicin.

Isolated from fungus and with the peptide sequence Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phl, alamethi-cin contains amino acids that are not part of the twenty main amino acids and their presence and the presence of two proline units, strongly induces an α-helix formation.

Theseα-helices which different from melittin, interact nicely with the lipid bilayer membrane; the channel formation, following a barrel-staves model, does

not disturb the membrane. It also requires the aggregation of around six units and leads to well-behaved channels in planar bilayer conductance experiments.

It also displays voltage dependance, which is attributed to conformational changes of the α-helix to expose a more important hydrophilic part, and a minor cation selectivity.^21,22

In both cases, even if a helical structure exist, it is not directly promoting transport by itself and the active barrel-staves model is strongly reminiscent of protein folding in the case of the anthrax toxin, for example. This is not necessarily the case and helical peptides can also promote ion transport by themselves. A good example for this is gramicidin A.

Gramicidin A, which is the major component of gramicidin D, an antibiotic produced by Bacillus brevis, has for its peptide sequence formyl-L-X-Gly-L- Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-ethanolamine with X being either Val or Ile.

Where the structure described for Alameticin contains uncommon amino acids, gramicidin alternates D and L amino-acids which allows the formation of a so-calledβ-helix. The active structure of gramicidin A has been a subject of discussion for a long time and the two proposed models are illustrated in Figure 3.^23–25

Figure 3:Crystal structures of gramicidin A (A: right-handed double helix from Cs⁺/methanol; B: left-handed double helix from ethanol) (left); Structure of gramicidin A in the membrane(right).²⁶

The main reason for this controversy is that, in solution, gramicidin A forms a double-stranded helical structure and its helicity depends on cation

1 Introduction 7

complexation. It is now known that the active structure of gramicidin A in the lipid bilayer membrane is a double single-strandedβ-helix with both formyl moieties in the middle of the membrane. Such a channel requires, therefore, the right arrangement of both parts of the active structure to be assembled.²⁷ The channel, which can be perceived as a longβ-helix, is sodium-selective, highly cation-selective, and can be blocked by the presence of divalent cations.

Several modifications of this channel have been reported.²⁶

Already at this point, the dimeric structure of gramicidin A can barely span the lipid membrane and smaller molecules would, most likely, fall into the category of carrier systems (or a higher degree of aggregation).

Natural carriers are somehow less common but several famous examples exist like valinomycin which is produced by many Streptomyces strains and is a cyclic dodecadepsipeptide with the formula [-L-Lac-L-Val-D-Hiv-D-Val-]3

(Figure 4). Along with those amide groups on the main chain, there are ester groups. The common point is that the twelve carboxylic oxygens are available for binding at the center of the cycle.²⁸

Given the geometrical constraints of the macrocycle, this carrier is potassium-selective. Binding to valinomycin is highly selective, displaying a difference as high as four orders of magnitude between its association constants for potas-sium and sodium.²⁹

HN

Figure 4:Chemical structures of valinomycin (left) and prodigiosin (right).

As previously mentioned, in order for the cation to travel through the mem-brane, the whole complex must shuttle through the membrane and this implies

that in most of the cases the process will be limited by the rate of diffusion.³⁰ Valinomycin is smaller than the aforementioned peptides and proteins, but it still has a considerable size. A better example of the reductionism that is possible in nature would beprodigiosin

Prodigiosin is part of the family of prodigiosines and is produced by Ser-ratia marcescens. It is a small non-peptidic anion carrier (Figure 4) that has been known for over one thousand years. Its transport mechanism is fairly well understood: coordination of chloride with the protonated form of prodi-giosin allows the symport transport of HCl. Recently, it has been shown that bicarbonate can be transported too, and thus prodigiosin can also act as a chloride/bicarbonate anion exchanger following an antiport mechanism. Given the importance of these anions, this is biologically relevant.³¹

Moreover, if prodigiosin research has developed in the last years, it is because it shows high activity as a drug, especially against cancer cell lines. This has somehow obscured the research of prodigiosin analogues as anion transport systems.^32–34Nevertheless, prodigiosin analogues have been studied and many structural variations are possible to improve its transport properties. This will be discussed in the following section.