Evolución de la Gestión del Recurso Humano

The dielectric barrier which exists between the intra and extra-cellular solution allows the free transport of uncharged molecules across the bilayer but actively prevents the passive transport of ions. This is due to the natural stability of charged molecules in the aqueous extra-cellular environment compared to the ‘oily’ intra-cellular solution (MacKinnon, 2004). Charged molecules are actively monitored by the cell by way of integral membrane proteins which selectively mediate their passage in and out of the cell. Ion channels do not transduce energy but function exclusively as selective pores whereas gradients are built by ion pumps which provide energy for transporters to carry out the housekeeping functions of the cell (Gouaux & MacKinnon, 2005).

1.3.1 Transporter architecture, selectivity and coupling

Translocation takes the form of three mechanisms, namely uniport, symport and antiport (Jessen-Marshall et al., 1995). Uniport refers to the facilitated diffusion of molecules by a channel or transporter one molecule at a time with a solute gradient. Symporters transport two different molecules in the same direction across a membrane, one against, one with its concentration gradient (Stryer, 1999). Finally, antiporters are like symporters though the two molecules are transported in opposing directions (Stryer, 1999). This dual movement allows one molecule to move with, and one move against a concentration gradient (Marger & Saier, 1993).

The α-helices of trans-membrane transporters (channels or pumps) are connected by a series of intra and extra-cellular loops. These are liable to vary in length and orientation to the membrane and may even be disjointed (Dutzler et al., 2002). Membrane proteins are not rigid in the bilayer, as flexibility is required for substrate binding and conformational changes in the transport process; this will be discussed later in this chapter. It is important to note this flexibility, as an assumption of a rigid protein may result in the misinterpretation of crucial structural data. Transport proteins are known to form dimers, trimers etc. where it is possible for each monomer to form or contribute to the formation of a translocation pathway. The overall architecture of membrane channels and transporters is highly variable, even between proteins which seemingly share function. Gouaux and MacKinnon have reviewed some of these structures which I have briefly summarised here (Gouaux & MacKinnon, 2005). Basin and hourglass structures allow the aqueous transport of substrate to the selectivity filter through the first envelope of the membrane as in the case of sodium dependant glutamate transporter (GltPh), and chloride channel (ClC), as discussed later. Pumps such as the Ca2+ ATPase and the GltPh transporter have no obvious pathways for the substrate to move toward or through. While channels such as the potassium (K+_{) channel are open to the continual} movement of ions, pumps require a conformational change to physically move the substrate across the membrane and thus have a lower through-put compared to ion channels.

The principle of selection, as regards membrane transporters, determines which ions are permitted to cross the membrane and which will be excluded. Major factors involved in ion selectivity are atomic composition and the stereochemistry of the binding site (Gouaux & MacKinnon, 2005). An ion must be physically detected, to be selected for or against; binding sites a priori must also be situated on the channel or pump at key locations to allow for substrate discrimination. When an ion is detected by a protein it is in a partially hydrated state as it is solubilised in the extra membrane solution. Following detection, and prior to translocation, the ion must be dehydrated to be translocated (Doyle et al., 1998; Gouaux & MacKinnon, 2005). Dehydration of a substrate is an energy demanding step and as such must be of ‘high value’ to the organism. Therefore, selection occurs when the energetic compensation is more favourable to one ion over the other, allowing for its discreet determination. When the dehydrated substrate enters the binding site the surrounding molecules interact with it, either directly or indirectly from the surrounding solution. For instance, in the leucine transporter, LeuT, leucine and sodium are co-transported with the Na+ gradient i.e. this process provides the energy for the transport of leucine. There are two binding sites in LeuT where five and six oxygen atoms (both full and partial charges) are in direct contact with the Na+ atom as it is transported (Yamashita et al., 2005). In the K+ channel, K+ _{is transported down an electrochemical gradient across four binding sites of the}

channel where the dehydrated K+ interacts exclusively with eight partial charges on the oxygen atoms of amino acids (Zhou et al., 2001). In this instance water is removed from the ions, then they are caged in counter charges, this region is known as the selectivity filter (Doyle et al., 1998). This shows that the protein selects for the particular cation by providing an oxygen-lined binding site of the appropriate cavity size for the trans-locating ion.

A feature of coupled transport systems is that the substrate is not permitted to collect within the receiving cells. Instead, it is quickly metabolised or stored for use later, though under controlled circumstances accumulation may be permitted (le Coutre & Kaback, 2000). Inbuilt ‘imperfections’ in biological systems can indirectly benefit organisms. For example, random mutation is the driving force behind natural selection and thus evolution. Likewise, the integral flaws of coupled transport can be beneficial to the cell. The stoichiometry of substrate and co-transporter(s) is system dependant. The stoichiometry of the mammalian divalent cation symporter, Dct1 (Nramp2), under normal conditions (pH 7) is 1:1 for Fe2+: H+, where the protons drive the transport of the cation. However, in conditions of high proton concentration (low pH) the stoichiometric ratio is not necessarily maintained and can be as much as 18 protons to every iron molecule, this is referred to as a ‘slip’, whereby the protons slip through the driving force pathway uncoupled (Nelson et al., 2002). Theoretically, at low pH, high proton concentration or negative membrane potential encourages the movement of excess protons across the transporter to no apparent detriment to substrate transport. This uncoupling can help to modulate the efficient functioning of the transport system and prevent the excessive accumulation of ions that may be damaging to the organism; this has been shown for other ions in addition to protons (Nelson et al., 2002). Channels can be opened either as a result of ligand binding (where the ligand does not cross the membrane) or they can be pH dependant, (where the proton is considered to be the ligand) (Perozo et al., 1999; le Coutre & Kaback, 2000). Ion channels although not involved with the transduction of energy can be involved with gating, whereby ions move with the concentration gradient as the gate is opened or closed (Miller, 2000). By contrast to the gating mechanism of the Major Facilitator Super-family (MFS), the machinery of the channel is abruptly altered in response to a stimulus (ligand binding or pH alteration) though the ‘rocker switch’ of the MFS is a slower fluid movement in answer to the proton electrochemical gradient (le Coutre & Kaback, 2000) (Figure 1.4).

A

B

C

D

Figure 1.4 The ‘alternating access’ model for membrane transport. The membrane protein is represented by yellow barrels and substrate molecules are represented as blue circles. A. The substrate is up-taken from either the periplasm or the cytoplasm. B. Substrate binding induces conformational changes within the protein. C. It is then moved through the bilayer and deposited in its destination. D. The protein returns to its open conformation.

Diagram based on that by Huang et al (2003)

In the case of permeases such as the lactose transporter (LacY) of E. coli, conformational changes are triggered in response to substrate binding (le Coutre & Kaback, 2000). With this substrate induced gating mechanism when substrate enters the binding site, protons on the other side of the membrane are already in position to be translocated. It is these protons which ‘hold’ the protein open to the extra-cellular environment (specific residues involved shall be discussed in Section 1.7) (le Coutre & Kaback, 2000; Sahin-Toth et al., 2000). While remaining in this conformation, the substrate becomes bound between two helices at the membrane surface; it is this binding which induces the conformational changes to translocate the substrate. As the substrate moves through the membrane the affinity of the permease is reduced and the substrate is released (protons are also released at this stage). This cycle returns to its ‘starting’ position, i.e. open to the extra-cellular solution when protons become bound again (Sahin-Toth et al., 2000).