Drag the different mechanisms of membrane transport to the appropriate figures.

13.1 Passive Diffusion

The simplest form of membrane transport, passive diffusion, refers to the diffusion of a species by random processes through the lipid bilayer of a membrane from one side of the membrane to another (and back again), independent of any metabolic energy. Net flux of the transported species in passive diffusion occurs only when there is a difference in the chemical potential of the species from one side of the membrane to the other side. Usually this chemical potential difference is the result of a difference in concentration of the species on one side versus the other. However, if the transported species is charged, the influence of a transmembrane electrical potential on the diffusion of the charged species can be considerable. Under passive diffusion, net movement of molecules can only occur down a concentration gradient, or from higher chemical potential (or chemical activity) to lower chemical potential.

The difference in chemical potential is directly related to the difference in activity of the chemical species on the two sides of the membrane.1 This can be expressed as

Δµi→j~lnajai

where ai and aj are the activities of the chemical species in question on either side of the membrane (Fig. 13.1). The greater the difference in activity of the species on the two sides of the membrane, the greater the difference in chemical potential of the species from one side of the membrane to the other. Therefore the greater the activity gradient across the membrane, the greater the driving force for the diffusion of the species in question across the membrane (and down a concentration gradient).

The flux of an uncharged species across a membrane is responsive to this activity gradient. The flux, or the number of molecules passing through the membrane per unit time in one direction, in the most simple case, can be thought of in the form of Fick’s law. This is the product of the activity gradient across the membrane of thickness x, daidx, and the diffusion coefficient:

Ji=−DKi daidx

where Ji is the flux of molecule i diffusing through the membrane, ai is the activity of molecule i, Di is the diffusion coefficient of molecule i, Ki is the partition coefficient of the diffusing species between membrane and water, and x is the bilayer thickness. As mentioned earlier, there can be an influence of a transmembrane electrical potential on the flux, if the diffusing species is charged. Thus if there is an electrical potential across the membrane, and the diffusing solute is charged, the electrical potential will play a role. The electrical potential will either increase the flux or decrease the flux, depending upon the charge on the diffusing solute and the disposition of the electrical potential.

The activity gradient refers to the activities of polar species in the aqueous domains on either side of the membrane. Nonpolar species can be expected to partition largely into the hydrophobic interior of the membrane, due to the hydrophobic effect, and to exhibit a negligible concentration in solution outside the membrane.

As a polar species transits a membrane, the most obvious barrier to its transit is the hydrophobic interior of the membrane. It is energetically unfavorable for the polar solute to partition into the hydrophobic membrane interior due to the hydrophobic effect. In the case of charged species, it has been estimated that the energy cost of introducing a small ion into the hydrophobic interior of the membrane is on the order of tens of kilocalories per mole.

How does a small, polar, uncharged solute transit the hydrophobic membrane interior? One likely mechanism involves the kinks that lipid hydrocarbon chains form under the influence of thermal motion. These kinks travel up and down the hydrocarbon chains of the lipids, as described in chapter “Lipid Dynamics in Membranes.” These kinks create dynamic packing defects in the bilayer structure that can be transiently occupied by small solutes, even polar solutes. The kinks can provide a pathway of small volume elements by which the polar, uncharged solute can cross the membrane, diffusing from defect to defect. This mechanism provides a good explanation for the transit of water across a membrane and the more limited diffusion of glucose across a lipid bilayer.

One consequence of this mechanism is that the larger the solute, the less favorable the fit between the solute and those volume elements. Thus the larger the polar species, the less effective such diffusion will be. For polar species with related structures, this expectation is fulfilled. For example, the permeability of lipid bilayers to sucrose is less than the permeability to glucose because of the larger size of the sucrose. Species carrying a formal charge are much less capable of entering the bilayer than polar, uncharged species because of the hydrophobic effect. The permeability of lipid bilayers to ions is consequently much less than to uncharged solutes. For example, the permeability of a lipid bilayer to sodium ion is very much less than the permeability of that same lipid bilayer to glucose.

The other consequence of this mechanism for polar solute diffusion across a lipid bilayer is that anything enhancing the occurrence of defects in the bilayer will enhance passive diffusion of polar solutes across the membrane. For example, lipid bilayers with more highly unsaturated lipids exhibit a higher incidence of packing defects among the lipid hydrocarbon chains in the bilayer interior (see chapter: Lipid Dynamics in Membranes). A higher incidence of packing defects results in an increase in membrane permeability.2 Even the reconstitution of membrane proteins into a lipid bilayer can increase membrane permeability (quite apart from any channels the protein might form by itself).3 Such an enhanced permeability may arise from packing defects at the lipid–protein interface due to transient mismatches between the rough protein surface and the lipid hydrocarbon chains.

The opposite effect is seen when perturbations to lipid bilayers lead to a decrease in packing defects. For example, a phase transition fully into a gel state will lead to tight packing of lipid hydrocarbon chains. Passive diffusion of small solutes across the membrane will become vanishingly small. Cholesterol alters the packing of lipid hydrocarbon chains in a membrane (see chapter: Cholesterol and Related Sterols: Roles in Membrane Structure and Function). Cholesterol reduces the incidence of kinks in the lipid hydrocarbon chains in the interior of the membrane. Therefore cholesterol reduces the probability of the formation of packing defects.4 Cholesterol consequently decreases passive permeability of lipid bilayers to small molecules like glucose. Cholesterol has the same effect in plasma membranes of mammalian cells, reducing passive permeability to solutes through the lipid bilayer and helping to seal the membrane.

Transit of the hydrophobic interior of the lipid bilayer is not the only barrier to passive diffusion through a membrane. Before encountering the lipid interior, a solute must get through an interfacial region on the surface of the membrane with considerably different properties than the bulk solution. This has been referred to as the unstirred layer between the lipid bilayer surface and the bulk water. The surface of the lipid bilayer is defined by the lipid headgroups interacting with the aqueous medium surrounding the membrane. Some water molecules are bound to the lipid headgroups. Furthermore there is a hydrodynamic effect due to the membrane surface. The water molecules near the surface, even those that are not bound, tend to experience a drag opposing their diffusion near the membrane surface. In addition, some lipid headgroups carry charges in their chemical structure. These charges are localized in the surface of the lipid bilayer. These charges are usually negative and attract positively charged counterions from the bulk aqueous environment toward the surface. This phenomenon creates a double layer (see chapter: Laboratory Membrane Systems) of oppositely charged species at the membrane surface, negative charges in the surface on the lipids and positive charges on counterions that form an array near the surface. This double layer can also create a barrier to the passive diffusion of solutes. Therefore, the surface of the membrane is a partially ordered array of solvent and solute molecules that extends for some distance from the surface. This rather special region presents a barrier through which a solute molecule must pass before encountering the interior of the lipid bilayer.

One other barrier to transport across the membrane is presented by the hydration of the species to be transported. Generally this hydration shell must be stripped away before the solute can enter the membrane lipid bilayer. Therefore, the energy of dehydration of the solute must be considered a barrier to the transport of that solute. Despite these barriers, solutes do manage to get across membranes.

So far this discussion has focused on the diffusion of polar, nonionizable solutes across lipid bilayers. Ionizable solutes sometimes have access to another important mechanism for transport across a lipid bilayer. For example, molecules with carboxylic acid moieties as part of an otherwise hydrophobic structure can exist in two distinctly different forms. In one form, the carboxyl is ionized and the molecule carries a net negative charge. This charged species does not effectively partition into the hydrophobic interior of the membrane (though it will orient in the surface of the membrane with the hydrophobic portion in the hydrophobic interior and the carboxyl in the surface with the polar lipid headgroups). However, the protonated molecule is reasonably nonpolar and its partition coefficient with respect to the hydrophobic interior is more favorable than that for the ionized species.

Therefore one mechanism for transport of ionizable organic acids across a membrane exploits the ability of the molecule to exist in two forms. The population of the molecules that is in the protonated form (governed by the pKa of the acid and pH of the solution) readily diffuses through the membrane. The dissociated population does not. However, even if there is only a very small percentage in the protonated form, depletion of that form by passage through the membrane is compensated by protonation of more solute molecules on the same side of the membrane. Thus movement of solute molecules through the membrane is achieved by funneling solute into the membrane permeable form and thence across the membrane. The more the pH of the medium and pKa of the ionizable group on the solute favor the permeable form, the greater will be the rate of transport of the species in question. Net flux across a membrane is only observed in the presence of a transmembrane gradient in the activity of the solute.

Abstract

Cystic fibrosis is the clinical example related to the principles explored in this chapter. Topics covered are types of membrane transport, exocytosis, facilitated diffusion, active transport requiring energy, simple and coupled transporters, ions and gradients, entry of magnesium and divalent ions into cells, proton transport, monocarboxylate symporter, citrate transporters in mitochondria and plasma membrane, intestinal transport of di- and tripeptides, amino acid transporters, glutamate synapse and excitatory amino acid transporters, fatty acid transport proteins, glycosylphosphatidylinositol anchor, voltage-gated sodium channels, epithelial sodium conductance channel, multidrug resistance channel, and blood–brain barrier. Closing the chapter is a summary, a list of references, an overview of multiple-choice questions, and a case-based problem.

4.5 Vectorial Metabolism, Group Translocation

Over 50 years ago, Peter Mitchell (see Chapter 18, Fig. 18.26) recognized the importance of what he termed “vectorial metabolism” [23,24]. Water-soluble enzymes convert substrate to product without any directionality. Mitchell proposed that many enzymes are integral membrane proteins that have a specific transmembrane orientation. When these enzymes convert substrate to product they do so in one direction only. This enzymatic conversion is therefore unidirectional, or “vectorial.” Mitchell expanded this basic concept into his now famous “chemiosmotic hypothesis” for ATP synthesis in oxidative phosphorylation (Chapter 18) [25,26]. For this revolutionary idea Mitchell was awarded the 1997 Nobel Prize in Chemistry.

Vectorial metabolism has been used to describe the mechanism for several membrane transport systems. For example, it has been reported in some cases the uptake of glucose into a cell may be faster if the external source of glucose is sucrose rather than free glucose. Through a vectorial transmembrane reaction, membrane-bound sucrase may convert external sucrose into internal glucose plus fructose more rapidly than the direct transport of free glucose through its transport system.

Mitchell defined one type of vectorial transport as group translocation, the best example being the PTS (phosphotransferase system) discovered by Saul Roseman in 1964.

PTS is a multicomponent active transport system that uses the energy of intracellular phosphoenol pyruvate (PEP) to take up extracellular sugars in bacteria. Transported sugars include glucose, mannose, fructose, and cellobiose. Components of the system include both plasma membrane and cytosolic enzymes. PEP is a high-energy phosphorylated compound (ΔG of hydrolysis is −61.9 kJ/mol) that drives the system. The high-energy phosphoryl group is transferred through an enzyme bucket brigade from PEP to glucose producing glucose-6-phosphate in several steps (PEP → EI → HPr →EIIA → EIIB → EIIC → glucose-6-phosphate). The sequence is depicted in more detail in Fig. 19.17 [27]. HPr stands for heat-stable protein that carries the high-energy ∼P from EI (enzyme-I) to EIIA. EIIA is specific for glucose and transfers ∼P to EIIB that sits next to the membrane where it takes glucose from the transmembrane EIIC and phosphorylates it producing glucose-6-phosphate. Although it is glucose that is being transported across the membrane, it never actually appears inside the cell as free glucose but rather as glucose-6-phosphate. Free glucose could leak back out of the cell via a glucose transporter, but glucose-6-phosphate is trapped inside the cell where it can rapidly be metabolized through glycolysis. Group translocation is defined by a transported solute appearing in a different form immediately after crossing the membrane.

Abstract

Membrane transport systems are crucial elements for plant nutrition and development as they play a key role in the absorption of mineral nutrients and water at the root level but also in the translocation within the plant. Moreover, membrane transport is involved in signalling and communication e.g. to adapt and interact with the environment. Most plants live in tight contact with beneficial soil microbes, such as bacteria and mycorrhizal fungi, which contribute to plant nutrition in part through modulation of the expression and functioning of plant transporter systems, as ion channels and transporters. In addition, mycorrhizal fungi largely increase the absorption surface of roots thereby promoting plant's access to soil resources as minerals and water. In turn, plants “reward” mycorrhizal fungi with sugars and/or lipids. This “fair trade” requires specific communication and a series of exchanges between the two symbiotic partners enabled by the adaptability and plasticity of their transporters. Here, we summarize recent advances allowing molecular insight in the impact of mycorrhizal symbiosis on the plant “transportome”. We highlight results obtained in ecto- and endomycorrhizal associations for plant transporters involved in the absorption of mineral nutrients and water released by the fungus at the symbiotic interface, and molecular players responsible for carbon and lipid nutrition of the fungal partner. We focus also on plant membrane transport systems implicated in early communication between plant and fungal partners.

Abstract

Membrane transport in fungi contributes to key aspects of their growth and development, and to their adaptation to multiple ever-changing environments. These transport systems, consisting of one or more proteins embedded in the cell membranes, enable the cell to ensure the uptake of essential nutrients and the efflux of toxic compounds, playing key roles in ion homeostasis and cell signaling. This article summarizes the general types of transport processes that enable translocation of substrates across fungal membranes and provide an overview of the transport systems characterized so far in arbuscular MYCO-Srrhizal fungi, the most ancient and widespread fungal plant symbionts.

Zinc Absorption in the Small Intestine and Zinc Transporter, ZIP4

Membrane transport of zinc does not require a redox reaction. This is sharply in contrast to those of iron and copper, which require the reduction to ferric iron or cupric copper, respectively, prior to uptake. Thus, the expression level of zinc transporters involved in zinc uptake in the enterocytes determines the net rate of zinc absorption. Vectorial transport of zinc into the enterocytes is primarily controlled by two zinc transporters, ZIP4 and ZNT1. ZIP4 is essential for uptake of dietary zinc into enterocytes, whereas ZNT1 is required for zinc efflux from the enterocytes into the portal vein (Fig. 9.1).28–30 Other zinc transporters are also expressed in the enterocytes, but their contribution to the absorption is thought to be minor.

Because apically localized ZIP4 plays a pivotal role as an entry route of dietary zinc from their lumens into enterocytes, mutations in the ZIP4/SLC39A4 gene result in a rare autosomal recessive genetic disorder of severe zinc deficiency, called acrodermatitis enteropathica (AE).31,32 Patients suffering from AE need daily oral zinc supplementation (1–3 mg/kg/day)33,34 for ameliorating zinc-deficient phenotypes, indicating that ZIP4 is the only zinc transporter indispensable for the uptake of zinc. The essentiality of ZIP4 in zinc absorption has been confirmed through enterocyte-specific deletion of the ZIP4 gene in mice, who cannot survive without zinc supplementation.35,36

The primary regulation of ZIP4 expression is conducted at the post-translational level, suggesting that food components may modulate its cell surface abundance through direct association. The ZIP4 protein is endocytosed and degraded in zinc-sufficient conditions, while it accumulates on apical membranes of the enterocytes during zinc deficiency.37–42 For example, ZIP4 in the duodenum of rats fed with a zinc-deficient diet (about 1/20th the amount of zinc found in a zinc-sufficient diet) was increased after only 1 day, and significantly increased after 2 days, whereas ZIP4 of rats fed a low-zinc diet (about 1/10th the amount found in a zinc-sufficient diet) was increased after 2 days and significantly increased within 4 days.43 Considering that overexpression of ZIP4 leads to increased zinc uptake into the cells and thereby enhances cellular zinc levels,44–46 food components with the ability to increase ZIP4 expression could potentially enhance zinc absorption, and thus be helpful in preventing zinc deficiency. To find such food compounds in diets, we established a novel screening strategy called “ZIP4 targeting,” using mouse Hepa cells, in which mouse ZIP4 (mZIP4) is expressed in a manner similar to that in the enterocytes. We also found that human AsPC1 cells could be used to evaluate the efficacy of food components to enhance ZIP4 abundance in human cells. To our knowledge, this is the first in vitro screening system to identify food components modulating cell surface ZIP4 abundance, which enabled us to investigate the efficacy of soybeans to improve zinc nutrition.

Abstract

The membrane transport apparatus is the most fundamental eukaryotic positioning system by which one-third of the mammalian proteome is processed and transported to the appropriate cellular destination or secreted. Over the last few years, secretory transport has been shown to be regulated by a network of signaling devices that sense and respond to both endogenous states and exogenous signals or perturbations in a harmonic and complex fashion, while robustly maintaining the homeostasis and optimality of the transport function. The design of this regulatory network can be best understood within the conceptual framework of the control theory.

Conclusions

Impaired membrane transport is now recognized as an important mechanism in the pathogenesis of DILI, notably DIC. Several hepatotoxic drugs have been shown to inhibit BSEP, although additional mechanisms such as mitochondrial toxicity and immune-mediated liver damage are usually required for clinically severe liver injury to occur. Of all the transporters characterized to date, BSEP is the best characterized in connection with a cholestatic pattern of injury. The p.V444A genetic variant, which is common in the population, appears to confer a risk for drug-induced and other forms of acquired cholestasis, although other BSEP variants have also been identified. The role of the MDR3 phospholipid translocator in DIC is still unclear. A functional in vitro assay for MDR3 transport activity is unavailable, thus making inhibition studies difficult. It seems likely that bile duct injury inflicted by drugs may in some cases be caused by inhibition of MDR3-mediated phospholipid excretion into the bile ducts, but this area of research remains a challenge for the future. In addition to the expression and genotype of transporters themselves, the intricate network of transcriptional and epigenetic regulatory mechanisms that affect transporter activity are under intense investigation, and new mechanisms by which drugs can adversely affect membrane transport are likely to emerge from this line of study. To date, functional testing of BSEP and MRP2 inhibition by drugs is commonly required by the US Food and Drug Administration in the context of liver signals during drug development, but interpretation of the results still requires an exact analysis of the individual clinical presentation in conjunction with the in vitro findings.

IV Outlook

Although intracellular membrane transport is a highly dynamic process, the importance of “still” images at high resolution as provided by EM is still invaluable. The introduction of new EM techniques, such as CLEM and electron tomography, especially in combination with immunolabeling techniques, will open up new avenues of research to gain even deeper insights into the mechanisms of transport. In this paper we have presented an overview of the most important techniques currently available for transport studies. We are convinced the use of live-cell imaging techniques in combination with high-end EM techniques, such as CLEM and tomography, will soon set the standard for membrane transport studies.

3.2.7.1 Event selection criteria

Flagellar membrane transport events are discriminated from nonspecific cell movement by the following selection criteria:

There must not be appreciable movement of either the flagellum or the cell body.

There must not be an appreciable difference in the level of the baseline before and after a transport event.

For events < 6 pN in magnitude where the flagellum is oriented such that it should have x- and y-axis components (i.e., the flagellum is oriented diagonally relative to the detector), then there must be movement evident in both channels and in the same direction (i.e., both anterograde or both retrograde) aligned to the flagellum.

For events < 6 pN which have the flagellum oriented along the x- or y-axis: there must be movement in the major axis channel aligned to the flagellum, but there does not need to be appreciable movement in the other channel.

For events > 6 pN in magnitude aligned to the flagellum, one may still observe small amounts of motion along the axis orthogonal to the flagellum. We do not exclude these events since neither the camera nor the flagellum is often perfectly aligned to the detector, accounting for what appear to be lateral deflections.