BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 5. BIOLOGICAL MEMBRANES

III. Transport of Substances Across Membranes

Any molecule can pass through a lipid bilayer; however, The rate of passive diffusion—i.e., the movement of a substance from a region of higher concentration to one of lower concentration—can vary significantly. For some molecules, this process takes so long that they are practically impermeable to the membrane lipid bilayer. The diffusion rate of substances across a membrane depends primarily on molecular size and relative lipid solubility.

Small nonpolar molecules such as O₂, Steroids, THYROID Hormones, and Fatty acids pass through the lipid membrane most easily via simple diffusion. Small polar uncharged molecules—such as СO₂, NH₃, Н₂O, ethanol, and urea—also diffuse at a fairly high rate. Glycerol diffuses considerably slower, while glucose is practically incapable of crossing the membrane independently. The lipid membrane is impermeable to all charged molecules, regardless of their size.

The transport of such molecules is made possible by Membrane Proteins that either form Water-filled channels (pores) in the lipid layer through which substances of a specific size can pass via simple diffusion, or by specific carrier proteins that selectively interact with certain ligands to facilitate their transmembrane transport (Facilitated Diffusion).

In addition to passive transport, Cells contain proteins that actively pump certain water-soluble substances against their concentration gradient—i.e., from a region of lower concentration to one of higher concentration. This process, known as Active Transport, is always mediated by carrier proteins and requires Energy Expenditure.

A. Structure and function of Protein Channels

Membrane channels are formed by integral proteins that span Cell/29.html">The Lipid Bilayer, creating a water-filled pore. The channel walls are lined with The amino acid side chains of these proteins.

Channels that differentiate substances solely by size, allowing all molecules below a certain threshold to pass along a concentration gradient (acting essentially as filters), are called "non-selective channels" or "pores". Such pores are found in the outer mitochondrial membrane, where porin protein molecules form wide hydrophilic channels. These channels permit the passage of all molecules with a molecular mass of 10 kD or less, including small proteins.

Selective channels are typically involved in the transport of specific ions. The ionic selectivity of these channels is determined by their diameter and The structure of their inner surface. For instance, cation-selective channels allow only cations to pass because they contain numerous negatively charged amino acid residues.

The opening or closing of selective channels is regulated either by Changes in the concentration of specific regulators—such as Neurotransmitters, hormones, Cyclic NUCLEOTIDES, NO, and G-proteins—or by changes in the transmembrane Electrochemical Potential (Fig. 5-14). The regulatory factor induces Conformational Changes in the channel-forming proteins, causing the channel to open and ions to flow down their concentration gradient. Transport through these channels does not induce conformational changes in the proteins themselves and depends solely on the concentration difference across the membrane. Consequently, the rate of transport through such channels can reach 10⁶–10⁸ ions per second.

Class="center">Fig. 5-14. A gated channel. Hatched squares represent regulators; light circles represent transported ions.

B. Facilitated Diffusion of Substances

Cell membranes contain translocase proteins. By interacting with a specific Ligand, they facilitate its diffusion (transport from a region of higher to lower concentration) across the membrane. Unlike protein channels, translocases undergo conformational changes during their interaction with the ligand and its subsequent transmembrane transfer. Kinetically, facilitated diffusion resembles an enzymatic reaction. Translocases exhibit a saturating ligand concentration at which all protein-ligand binding sites are occupied, allowing the proteins to operate at maximum velocity (V_max). Therefore, the rate of facilitated transport depends not only on the concentration gradient of the transported ligand, but also on the number of carrier proteins in the membrane.

Some translocases transport only a single water-soluble substance from one side of the membrane to the other. This type of simple transport is called "passive uniport". An example of uniport is the function of GLUT-1, a translocase that transports glucose across The erythrocyte membrane (Fig. 5-15):

Fig. 5-15. Facilitated diffusion (uniport) of glucose into erythrocytes via GLUT-1 (S — glucose molecule). The glucose molecule binds to the carrier on the outer surface of The Plasma membrane. A conformational change occurs, exposing the carrier's binding site—now occupied by glucose—to The Cell interior. As a result of these conformational changes, the carrier loses its affinity for glucose, and the molecule is released into the cell Cytosol. The dissociation of glucose triggers a conformational change in the protein, returning it to its original conformation.

Some translocases can transport two different substances down their concentration gradients in the same direction (passive symport) or in opposite directions (passive antiport) (Fig. 5-16).

Fig. 5-16. Types (modes) of carrier-mediated (translocase-mediated) facilitated diffusion. S₁ and S₂ represent different molecules.

An example of a translocase operating via passive antiport is the anion exchanger of the erythrocyte membrane (Fig. 5-17).

Fig. 5-17. Passive antiport of НСO₃^- and Cl^- anions across the erythrocyte membrane. A — when an erythrocyte is in venous capillaries, НСO₃^- ions generated by carbonic acid dissociation move out into the Blood along their concentration gradient. In exchange for each НСO₃^- ion transported out of the cell, the translocase imports a Cl^- ion; B — when the blood reaches the Lungs, the translocase reverses the direction of Ion Exchange. This "shuttle" system operates very rapidly, ensuring the removal of СO₂ from the body while maintaining an optimal intracellular pH.

The inner mitochondrial membrane contains numerous translocases that mediate passive antiport (Fig. 5-18). This transport process involves an equivalent exchange of ions, though not always an equivalent charge exchange.

Fig. 5-18. Selected mitochondrial transporters.

B. Structure and function of carrier proteins mediating active transport

The transport of certain ligands (ions, glucose, Amino Acids) across membranes occurs against a concentration gradient and requires energy expenditure (active transport). Ligand Transport Across the membrane driven by ATP energy expenditure is referred to as "primary active transport."

1. Primary active transport

The transport of certain inorganic ions proceeds against their concentration gradient with the participation of transport ATPases (ion pumps). All ion pumps simultaneously function as Enzymes capable of autophosphorylation and autodephosphorylation. ATPases differ in their ion Specificity, the number of ions transported, and the direction of transport. As a result of ATPase activity, the transported ions accumulate on one side of the membrane. The most widespread ATPases in the plasma membrane of human cells are the Na⁺, K⁺-ATPase, Ca²⁺-ATPase, and H⁺, K⁺-ATPase of the gastric mucosa.

Na⁺, K⁺-ATPase

This carrier enzyme catalyzes the ATP-dependent transport of Na⁺ and K⁺ ions across the plasma membrane. The Na⁺, K⁺-ATPase consists of α and β subunits; α is the large catalytic subunit, and β is the small subunit (a glycoprotein). The active form of the translocase is a tetramer (αβ)₂ (Fig. 5-19).

Fig. 5-19. Structure and function of the plasma membrane Na⁺, K⁺-ATPase. 1 - three sodium ions bind to specific sites on the translocase; 2 - a conformational change of the translocase, induced by the binding of 3Na⁺, leads to the activation of the catalytic subunit and an increased affinity of the Active Site for the substrate (ATP). An autophosphorylation reaction occurs at the carboxyl group of an aspartic acid residue; 3 - autophosphorylation alters the charge and conformation of the translocase, causing it to close on the inner side of the membrane and open to the outside, decreasing the affinity for sodium ions which then dissociate from the carrier; 4 - the Na⁺, K⁺-ATPase, now open to the extracellular side of the membrane, features a specific binding site for 2K⁺; the binding of two potassium ions to the phosphorylated translocase induces a conformational change and the appearance of autophosphatase activity, and an autodephosphorylation reaction takes place; 5 - dephosphorylation alters the charge and conformation of the translocase, closing it on the outer side of the membrane and opening it to the inside, decreasing the affinity for potassium ions, which then dissociate from the Na⁺, K⁺-ATPase; 6 - the ATPase returns to its initial state.

The Na⁺, K⁺-ATPase is responsible for maintaining a high intracellular concentration of K⁺ and a low concentration of Na⁺. Because the Na⁺, K⁺-ATPase pumps out three positively charged ions while pumping in two, an electrical potential is generated across the membrane, with the inside of the cell being negative relative to its outer surface.

Ca²⁺-ATPase

In the cytosol of "resting" cells, the Ca²⁺ concentration is ~10^-7 mol/L, whereas outside the cell it is ~2 • 10^-3 mol/L. This concentration gradient is maintained by the active calcium ion transport system, whose main components are calcium pumps—Ca²⁺-ATPases and Na⁺, Ca²⁺ exchangers.

The Ca²⁺-ATPase is localized not only in the plasma membrane but also in The Endoplasmic reticulum (ER) membrane. The enzyme consists of ten transmembrane domains spanning The cell membrane. Between the second and third domains lie several aspartic acid residues involved in calcium binding. The region between the fourth and fifth domains contains the site for ATP attachment and autophosphorylation at an aspartic acid residue. The plasma membrane Ca²⁺-ATPases of certain cells are regulated by the protein calmodulin. Each of the Ca²⁺-ATPases of the plasma membrane and ER exists in several isoforms.

The stages of cytoplasmic membrane Ca²⁺-ATPase function are illustrated in Fig. 5-20.

Fig. 5-20. Sequence of events during Ca²⁺-ATPase operation. 1 - binding of two Calcium Ions to the cytosolic-facing region of the ATPase; 2 - A change in the charge and conformation of the enzyme (ATPase) induced by the binding of two Ca²⁺ ions, leading to an increased affinity for ATP and activation of autophosphorylation; 3 - autophosphorylation is accompanied by conformational changes, causing the ATPase to close on the inner side of the membrane and open to the outer side; 4 - a decrease in the affinity of the binding sites for calcium ions occurs, and they dissociate from the ATPase; 5 - autodephosphorylation is activated by magnesium ions, resulting in the Ca²⁺-ATPase losing its phosphate group and two Mg²⁺ ions; 6 - the ATPase returns to its initial state.

Impairment of Ca²⁺-ATPase activity in pathology. One of the causes of impaired Ca²⁺-ATPase function is the activation of membrane Lipid Peroxidation (LPO). Both fatty acid acyl residues within Phospholipids and SH-groups in the Active Site of the enzyme undergo oxidation. Disruption of the lipid microenvironment and the STRUCTURE OF THE active site leads to a conformational change in the ATPase, a loss of affinity for calcium ions, and a diminished capacity for autophosphorylation. The ATPase ceases to pump calcium ions out of the cell cytosol, leading to an elevated intracellular calcium concentration. Ca²⁺ enhances Muscle contraction, increases vascular wall tone, and results in elevated blood pressure. Impaired Ca²⁺-ATPase function also plays a significant role in The Development of atherosclerosis, Cancer, and immune pathologies.

2. Secondary active transport

The transport of certain soluble substances against their concentration gradient relies on the simultaneous or sequential transport of another substance down its concentration gradient in the same direction (active symport) or in the opposite direction (active antiport). In human cells, the ion transported down its concentration gradient is most frequently Na⁺.

An example of this type of transport is the plasma membrane Na⁺, Ca²⁺ exchanger (active antiport), where sodium ions are transported into the cell down their concentration gradient while Ca²⁺ ions are extruded from the cell against their concentration gradient (Fig. 5-21).

Fig. 5-21. Sodium-dependent calcium ion transport. A - Na⁺-dependent calcium ion carrier; B - Na⁺, K⁺-ATPase.

Active symport is the mechanism responsible for the intestinal absorption of glucose and the reabsorption of glucose and amino acids from the primary urine by renal tubular cells (Fig. 5-22).

Fig. 5-22. Mechanism of active symport. A - Na⁺ and glucose bind to different sites of the translocase. The ions tend to enter the cell down their concentration gradient and "drag" glucose along with them; if the extracellular concentration of Na⁺ decreases, Glucose Transport into the cells drops; B - sodium ions entering the cell together with glucose are "pumping out" back by the Na⁺, K⁺-ATPase, which maintains the Na⁺ concentration gradient and controls glucose transport.

G. Transmembrane Transport of Macromolecules and particles: Endocytosis and Exocytosis

Transport proteins facilitate the movement of small polar molecules across the cell membrane, but they are unable to transport macromolecules, such as proteins, Nucleic Acids, Polysaccharides, or even larger particles. The mechanisms by which cells ingest such substances or expel them from the cell differ from the transport mechanisms of ions and polar compounds.

Endocytosis

The process by which a substance is transferred from the extracellular environment into the cell along with a portion of the plasma membrane is called endocytosis. Via endocytosis (phagocytosis), cells can engulf large particles such as Viruses, Bacteria, or cell debris. The uptake of large particles is mainly carried out by specialized cells known as phagocytes.

The uptake of fluid and dissolved solutes via small vesicles is called pinocytosis. The uptake of substances via endocytosis (pinocytosis) is characteristic of all cells.

The endocytosis cycle begins in specific Regions of the plasma membrane called coated pits (Fig. 5-23). Coated pits account for only 1–2% of the total membrane surface area. The protein clathrin forms lattice-like structures associated with depressions on the plasma membrane surface.

Fig. 5-23. Sequence of events in The formation of a coated vesicle from a coated pit.

Coated pits invaginate into the cell, constrict at their base, and pinch off from the membrane to form coated vesicles (pinocytic vesicles). The lifespan of coated pits is short; they form within a minute and then undergo the endocytosis cycle.

Substances contained within pinocytic vesicles do not mix with other cellular macromolecules. Their pathway ends in Lysosomes, whereas the clathrin-containing membrane Components of the vesicles are recycled back to the plasma membrane.

Receptor-mediated endocytosis, which takes place with the participation of receptors embedded in coated pits, allows cells to internalize specific substances. Macromolecules or particles are bound by receptors and accumulate in the coated pit. This is followed by invagination into the cell and the pinching off of an endocytic vesicle containing the ingested substance, the membrane components of the coated pit, and the receptor. Different coated pits may incorporate different receptors.

An example of receptor-mediated endocytosis is the uptake of Cholesterol into The Cell as part of low-density Lipoproteins (LDLs) (Fig. 5-24).

Fig. 5-24. Localization of LDL receptors in the cytoplasmic membrane. A - localization of LDL receptors within a coated pit; B - localization of defective LDL receptors outside the coated pit.

The number of receptors in the plasma membrane coated pit varies depending on the cell's demand for cholesterol. Mutations in the Gene encoding the LDL receptor impair its structure, preventing it from localizing to the coated pit region of the plasma membrane. While the localization of the receptor outside the coated pit does not reduce its complementarity to LDL, the endocytosis of the receptor-LDL complex fails to occur.

Exocytosis

Macromolecules, such as Plasma Proteins, Peptide Hormones, digestive enzymes, Extracellular matrix proteins, and lipoprotein complexes, are synthesized within cells and subsequently secreted into the extracellular space or blood. Because the membrane is impermeable to such macromolecules or complexes, their secretion occurs via exocytosis. A key feature of exocytosis is that the secreted substances are sequestered within vesicles and do not mix with other cellular macromolecules or Organelles. During exocytosis, the contents of secretory vesicles are released into the extracellular space when the vesicles fuse with the plasma membrane.

Both regulated and constitutive pathways of exocytosis exist in the body. Constitutive secretion is characterized by the continuous synthesis of secretory proteins, their packaging into transport vesicles in the Golgi apparatus, and their delivery to the plasma membrane for secretion. An example is the Synthesis and Secretion of Collagen by fibroblasts to form the extracellular matrix.

Regulated secretion involves the storage of export-ready molecules in transport vesicles and their fusion with the plasma membrane only upon exposure of the cell to a specific stimulus. Regulated secretion accounts for the release of digestive enzymes during Digestion, as well as the secretion of hormones, neurotransmitters, and other BIOLOGICALLY ACTIVE SUBSTANCES. A prime example of this secretion type is the release of the peptide hormone Insulin into the bloodstream after a meal. The stimulus for the secretion of insulin—stored in secretory granules of the pancreatic β-Cells of the islets of Langerhans—is an increase in glucose concentration in the blood and within the β-cells (Fig. 5-25).

Fig. 5-25. Regulation of Insulin secretion. An increase in glucose concentration leads to an elevated ATP/ADP ratio in the β-cell, closure of ATP-sensitive potassium channels, membrane depolarization, and opening of voltage-gated calcium channels. Elevated intracellular concentrations of potassium and calcium ions in the β-cell trigger the fusion of secretory vesicles (insulin-containing granules) with the membrane and the release of vesicle contents (insulin) from the cell.