BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 4. MOLECULAR ORGANIZATION AND BIOLOGICAL FUNCTIONS OF MEMBRANES

4.3. Membrane Functions

4.3.1. Membrane Transport

Living systems at all Levels of Organization, including Cells and Cell Organelles, are open thermodynamic systems. Therefore, membrane Transport of substances is a prerequisite for life. Impairments in substance transport across Biomembranes lead to pathology. In fact, medical Treatment itself is largely associated with the interaction and transfer of therapeutic agents across membranes, either directly or indirectly via Liposomes. The transport of substances across cell membranes can be divided into two types: active and passive (Fig. 4.8).

Class="center">

Fig. 4.8. Schematic representation of the MAIN TYPES OF Transmembrane Movement of substances

Naturally, the better substances dissolve in Lipids, the easier they cross the membrane—a process known as diffusion. Polar substances pass through the membrane with greater difficulty because they are insoluble in lipids. At the same time, Water penetrates well (its P = 10^-2 cm/s). This is explained not only by the fact that water is transported through Ion Channels and lipid pores (see below), but also by the fact that it is transported via kinks—free cavities between phospholipid tails (kinks) formed during their thermal motion. These cavities carry small hydrophilic molecules across the membrane, primarily water molecules that have entered them.

Facilitated Diffusion occurs with the participation of carrier molecules. An example is the antibiotic valinomycin, a carrier of K⁺ ions. It is a cyclic depsipeptide with a Molecular Weight of 1111: L-lactate-L-valine-D-oxyisovaleric acid-D-valine. A distinctive feature of valinomycin's chemical Structure is that its polar groups are localized inside the structure, while nonpolar hydrophobic residues of valine molecules are localized on the outside. Such a structure allows it to form a complex with potassium ions that is soluble (like free valinomycin) in the lipid phase of the membrane. Therefore, these complexes are "captured" by the membrane, where they diffuse down the concentration gradient

and transport potassium to the other side of the membrane. Valinomycin, now free of potassium, returns back (also down its concentration gradient) and complexes with a new K⁺ ion (Fig. 4.9). Thus, carrier-mediated shuttling of potassium ions across the membrane takes place.

Another type of facilitated diffusion is channel-mediated ion transport. An example of this is the gramicidin channel shown in Fig. 4.9, B. Molecules of this antibiotic easily integrate into the membrane (due to its Hydrophobic surface) and, by dimerizing at their N-termini, form a channel that allows cations to pass in a Relay-like manner without changing its position within the membrane. These Two Types of substances are referred to as ionophores and channel formers, respectively.

Fig. 4.9. Diagram of induced ion transport:

A - by the ionophore valinomycin and B - by the channel former gramicidin.

B - STRUCTURE OF THE K⁺-valinomycin complex: the K⁺ ion is fixed in the center due to ion-dipole interaction involving the carbonyl groups of the peptide

Thus, the differences between facilitated and simple diffusion are as follows:

✵ the transport of substances with the participation of carriers proceeds faster;

✵ facilitated diffusion exhibits saturation kinetics (when all carrier molecules are occupied by the substance, further increasing its concentration does not increase the substance flux rate) (Fig. 4.10);

✵ there are substances that block facilitated diffusion—inhibitors—by forming a stable complex with carrier molecules.

Fig. 4.10. Kinetics of simple (1) and facilitated (2) diffusion:

A: 1 - velocity is proportional to the concentration of the substance moving across the membrane; 2 - velocity reaches a maximum value (Vmax) upon saturation of carrier Proteins. The binding constant of the substance to the carrier (Km) is the concentration of the substance at which ν = 1/2 Vmax. [S] - concentration of the substance being transported across the membrane.

B - curve 2 in Lineweaver-Burk coordinates, which allow the Determination of the main kinetic parameters of the transport process: the Michaelis constant Km and the maximum process velocity Vmax

If a solute molecule is uncharged, the direction of its transmembrane movement is determined solely by the concentration difference (concentration gradient) of this substance on both sides of the membrane—the chemical potential. If the molecule carries an electrical charge, its transmembrane movement is influenced not only by the chemical potential, but also by the electrical potential difference across the membrane (Membrane Potential, see below)—the electrical potential. Together, the chemical and electrical potentials constitute the Electrochemical Potential, μ.

Some carrier proteins transport a single substance across the membrane—such transport is called uniport. Other proteins act as cotransport systems, where the transport of one substance depends on the transport of another. If these two substances are transported in the same direction, it is symport; if in opposite directions, it is antiport. An example of uniport is the transport of glucose into a Muscle cell (where its concentration is always lower than in the extracellular space). An example of symport is the uptake of glucose by intestinal epithelial cells along with Na⁺ ions, whose extracellular concentration in the extracellular space is high. An example of antiport is the transport of Cl^- and HCO₃^- ions by the erythrocyte band 3 protein and the transport of Na⁺ and Ca²⁺ by the sodium-calcium exchanger in muscle and Nerve Cells.

The MOLECULAR MECHANISMS OF carrier proteins involve reversible Conformational Changes in their molecules (Fig. 4.11). These conformational shifts ensure the binding of substances on one side of the membrane and their release on the other side. As can be seen from Fig. 4.11, a carrier protein exists in three conformational states: A – open on the outside ("pong" state); B – intermediate, loaded with glucose; C – open on the cytoplasmic side ("ping" state). This "ping-pong" system facilitates the transport of glucose from the extracellular environment, where its concentration is higher. The dashed lines indicate the Na⁺ ion binding sites. Driven by the sodium concentration gradient, The rate of glucose transport increases—a process known as symport.

Fig. 4.11. Scheme illustrating conformational changes in a carrier protein mediating glucose uniport across the membrane.

Symport – transport of glucose coupled with Na⁺ ions

Channels and pores (Table 4.2) also undergo conformational changes in their protein subunits, but these changes merely regulate their opening and closing rather than directly effecting translocation. Based on their regulation mechanism, channels are classified into voltage-gated and chemically gated (Ligand-gated). The former respond to changes in transmembrane potential, while the latter open in response to specific chemical agents (Neurotransmitters). The terms "pore" and "channel" are often used interchangeably, but they are distinct. A pore is a non-selective Membrane Structure that "recognizes" substances primarily by molecular size. Pores do not change their conformation during transport and thus allow only small molecules to pass. Channels refer to ion channels that selectively transport major "physiological" cations, hence they are designated as K⁺, Na⁺, Ca²⁺, and H⁺ channels. In other words, a pore is a constitutively open structure, whereas a channel is a dynamic structure that changes conformation, "breathes," and can exist in at least three states: closed, intermediate, and open.

Table 4.2

Classification of Membrane Transport Systems

Osmosis as a type of passive transport is essentially the Diffusion of Water from regions of higher concentration to regions of lower concentration (or the net Movement of water toward higher solute concentrations). Osmosis is a critically important phenomenon in many biological processes, including the hemolysis of erythrocytes in hypotonic solutions and turgor in plants.

Active Transport maintains concentration gradients of substances, electrical potentials, and pressure differences within organisms. From a thermodynamic perspective, active translocation of substances and ions keeps the Organism in a non-equilibrium state (equilibrium means death for an organism). Biological

membranes contain several types of ion pumps that operate using energy derived from ATP Hydrolysis. There are three main types of electrogenic pumps (Fig. 4.12), which are based on transport ATPases. During the operation of the Na⁺,K⁺-ATPase, the energy from the high-energy bond of a single ATP molecule drives the export of three Na⁺ ions out of The Cell and the import of two K⁺ ions into the cell. This establishes K⁺ and Na⁺ concentration gradients across The Plasma Membrane, directed inward (for potassium) and outward (for sodium). Because one more ion is pumped out than is pumped in per ATP molecule (1 ATP : 2 K⁺ : 3 Na⁺), a membrane potential is generated, known as the resting membrane potential (RMP), with a negative sign on the cytoplasmic side (see 4.3.2). The Ca²⁺-ATPase drives The active transport of two Ca²⁺ ions into the cisternae of The Endoplasmic reticulum, while the proton pump (H⁺-ATPase) transports two protons per ATP molecule (Fig. 4.12). The Molecular Mechanism of this transport in the case of the sodium pump involves seven stages of ion translocation coupled with ATP hydrolysis. As shown in the diagram in Fig. 4.12B, the key steps of enzyme action are: 1) formation of an enzyme-ATP complex on the cytoplasmic side, activated by Mg²⁺ ions; 2) binding of three Na⁺ ions by the complex; 3) phosphorylation of the enzyme resulting in ADP formation; 4) "translocation" (flip-flop) of the enzyme with sodium ions across the membrane interior; 5) ion-exchange reaction substituting sodium for potassium on the outer surface of the plasma membrane; 6) reverse translocation of the enzyme complex carrying two potassium ions into the cell; 7) return of the enzyme to its initial state with the release of potassium ions and inorganic phosphate.

The active transport described above is classified as primary. There are also secondary active transport systems for substances that lack dedicated pumps (such as CARBOHYDRATES and Amino Acids). In this case, substance transport is mediated by the membrane potential and/or ion concentration gradients via specific carrier proteins embedded in the membrane. There are several types of secondary active transport. One of them is the transport of monovalent ions via a carrier that crosses the membrane equally well in both its "loaded" and empty states. The energy source here is the RMP. Unidirectional transport mediated by a specific carrier is termed (as in facilitated diffusion) uniport. The net result of this transport is the accumulation of ions driven by the dissipation of the membrane potential. An example of uniport is the accumulation of K⁺ ions in mitochondria in the presence of valinomycin (see Fig. 4.9).

Fig. 4.12. Scheme of Active ion transport by transport ATPases (A) and the stages of this transport for Na⁺,K⁺-ATPase (B): E – ATPase enzyme on the inner (E₁) and outer (E₂) surfaces of the plasma membrane;

P – inorganic phosphate in the reaction ATP → ADP + P; * – active complex.

C – scheme of Na⁺,K⁺-ATPase localization within the membrane as a tetramer: α and β subunits of the enzyme

The opposite flux of ions mediated by carrier molecules is called antiport. Translocation occurs in two steps: first, one ion crosses the membrane from left to right, and then the second ion moves in the opposite direction (Fig. 4.13). Importantly, the membrane potential remains unchanged during this process, and the driving force for translocation is the concentration gradient of one of the ions established by primary active transport. A classical example of antiport is the transmembrane exchange of potassium and hydrogen ions facilitated by the antibiotic nigericin.

Fig. 4.13. Schematics of the three main types of secondary active ion transport:

A – uniport; B – antiport; C – symport

In symport, two electroneutral entities reside in the membrane: a carrier complexed with a cation and an anion, and an empty carrier (Fig. 4.13C). Since the membrane potential does not change according to this scheme, the driving force for transport is the concentration gradient of one of the ions. It is widely accepted that Amino acids are accumulated by cells via a symport mechanism. The sodium-potassium pump establishes an initial sodium ion concentration gradient (Fig. 4.12A), which subsequently drives the accumulation of amino acids into the cell via symport. As indicated by the symport scheme, the process must be accompanied by significant shifts in osmotic equilibrium, as two particles are transported across the membrane in the same direction during a single cycle.

A few caveats should be noted regarding the classification of membrane transport types. Such classifications, based on both structural features and Energy Requirements, are somewhat conventional. For instance, terms such as carriers, translocases, and permeases—frequently used across various textbooks in reference to transport proteins—are largely synonymous. Typically, the term "permease" is applied when describing bacterial transport proteins; "carrier" is more commonly used for ionophores and carriers in eukaryotic Plasma Membranes; and "translocase" is used in discussions of general Transmembrane Translocation phenomena. The categorization of membrane transport processes involves even more conventions. In truth, no transmembrane transport occurs without the expenditure of energy. The translocation of ions via ionophores and channel formers (discussed above) is only possible when an ion concentration gradient exists. Under normal conditions, however, such gradients are only generated through the consumption of energy. Therefore, in our example (Fig. 4.9), the transport of K⁺ ions by valinomycin (uniport) is only possible after the Na⁺,K⁺-ATPase has established a potassium concentration gradient using energy from ATP hydrolysis. Similarly, Amino Acid Transport driven by the dissipation of a Na⁺ ion gradient (symport) cannot occur until the cell establishes that gradient using ATP hydrolysis energy via a membrane ATPase. Antiport is likewise a process that increases the gradient of one ion (or substance) at the expense of dissipating the gradient of another, which serves as the driving force for antiport (Fig. 4.13). Even passive diffusion processes across membranes are, broadly speaking, energetically demanding: the direction of diffusive transport is determined by the concentration gradient of the substance, the creation of which requires Energy Expenditure.

In animal cells, the co-transported ion is typically Na⁺. Its electrochemical gradient provides the energy for the active transport of another ion or a neutral molecule. Each membrane transport system (protein components) is specific for the cellular uptake of a small group of related sugars or amino acids (which is clearly evident when examining their absorption by intestinal epithelial cells). In such systems, these molecules and Na⁺ ions bind to distinct sites on the carrier protein (Fig. 4.10): sodium ions tend to enter the cell down their concentration gradient and "drag" glucose (or amino acids) along in the same direction. The steeper the Na⁺ gradient, the higher the transport rate.

In plants and animals, H⁺ is often utilized as the co-transported ion. A prime example is the lactose permease carrier. This is a transmembrane protein consisting of a single polypeptide chain that spans the membrane nine times. For every molecule of lactose brought into the cell, one proton is co-transported, with its gradient maintained by an H⁺-ATPase.

Constant intracellular pH (7.1–7.2) is maintained in all cells. This is primarily achieved via the Na⁺,H⁺ exchanger (antiport): excess protons continuously generated during redox reactions are expelled in exchange for incoming extracellular Na⁺ ions. This exchanger-carrier is activated by low pH values, driven by H⁺ binding on the cytoplasmic face of the membrane. Conversely, another exchanger is activated by high pH (~7.7)—the Cl^-,HCO₃^- antiport: the efflux of HCO₃^- in exchange for Cl^- lowers the cytosolic pH.

When studying membrane transport, one cannot ignore how a cell "prevents" a substance from leaving via the exact same pathway it entered. Such mechanisms do exist. For instance, glucose transported into a cell can be modified or transported out to another "required" destination. The latter method is transcytosis, the transport of glucose from intestinal epithelial cells into the bloodstream. During transcytosis, glucose enters the cell via a K⁺-dependent symport localized in the apical region of the plasma membrane, and exits the cell down its concentration gradient via a uniporter located in the basolateral membrane. Tight junctions prevent the diffusion of symporter and uniporter carrier systems across the entire plasma membrane. Consequently, carrier proteins are confined to their respective membrane domains (membrane Asymmetry) and drive transcytosis. However, a fraction of glucose in such epithelial cells is not transported into the Blood via transcytosis, but is instead utilized for the cell's own energy needs. To this end, glucose is phosphorylated by glucose-6-phosphatase (with ATP serving as the phosphate donor) to yield glucose-6-phosphate. Since this is now a charged molecule, it cannot escape and thus enters glycolytic pathways. In bacteria, an analogous system uses phosphoenolpyruvate as the high-energy phosphate donor. Because phosphate groups are transferred to the glucose molecule only after entering the cell, this type of transport is sometimes referred to as vector or directed group translocation.

Thus, throughout the cell's metabolic activity, various substances cross its plasma membrane in a highly regulated and controlled manner. This is accomplished by membrane transport systems, which comprise ion pumps, carrier molecules, and highly selective channels.