BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 4. MOLECULAR ORGANIZATION AND BIOLOGICAL FUNCTIONS OF MEMBRANES

4.3. Membrane Functions

4.3.2. Pores, Channels, and Electrogenesis

Pores in Introduction/36.html">Biological Membranes are formed by Proteins and Lipids. Water-filled protein pores are most commonly found in the outer membranes of Bacteria and Cell/35.html">Mitochondria and are composed of the protein porin and similar proteins. In the Plasma Membranes of animal Cells, pores are lipid in nature. Gap Junctions between cells also have a pore-like Structure. These structures (pores) vary in size and are non-specific to the substances passing through them. In contrast, protein channels are smaller than protein pores and primarily serve for the specific transport of ions. They are called Ion Channels.

Porins form pores that function as molecular sieves, allowing the diffusion of small (up to 600 Da) molecules across the membrane (Fig. 4.14). More than 40 porins with a molecular mass of 28-48 kDa are known. Their main feature is that they form a transmembrane channel: β-chains form a β-barrel, the inner walls of which are lined with polar charged amino acid residues. Porin pores vary greatly in size (0.6-23 nm); their trimers form three independent channels (Fig. 4.14, A) or one large channel, and the "matrix porin" from E. coli has three inlets on the outer side of The Plasma Membrane, which then merge to form a single exit into the periplasm. Outer mitochondrial membrane porins also form a cylindrical structure of β-structural elements, which acts as a voltage-dependent anion-selective channel.

As in any crystal, various defects occur in the phospholipid bilayer, among which the lipid hydrophilic pore defect is of major importance (Fig. 4.14, B). As shown in the figure, the pore has two radii: d/2 and r. Two opposing forces act at the boundary of the pore: one, the edge line tension of the pore perimeter (d/2), promotes pore growth, while the other, the surface tension of the bilayer (r), causes the pore to contract. An unstable state of the membrane occurs at a critical radius value - r*: when r > r*, the membrane ruptures; when r < r*, the pore "heals" (closes), and the membrane remains stable (self-heals). The critical radius of lipid pores in a liquid-crystalline membrane reaches 9 nm.

Since the resting Membrane Potential (see below) does not exceed 0.1 V (0.2 V in mitochondria) and the membrane thickness does not exceed 10 nm, the electric field strength across the membrane reaches 10 ⁷ V/m. The membrane is a more perfect electrical insulator than many technical insulators. Therefore, electrical breakdown by its own RMP is unlikely. However, the RMP can increase due to an external electric field and exceed the threshold RMP values for electrical breakdown. The result of the latter is The formation of lipid pores. This phenomenon is also used in biotechnology: to increase membrane porosity (electroporation), introduce DNA into The Cell (electrotransfection), deplete the cell of large molecules (electropermeabilization), fuse cells (electrofusion), and also to explain the hemolysis of erythrocytes in cryobiology.

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Fig. 4.14. Diagram of membrane hydrophilic pores:

A - made of porin (top view): 1, 2, 3 - three independent channels formed by a porin trimer;

4 - β-chain of porin with hydrophilic (blue) and hydrophobic (red) amino acid residues.

B - made of lipids (cross-section): d - lipid bilayer thickness; d/2 - radius of wall curvature; r - pore radius.

C - cross-section of a nuclear pore with a 9 nm diameter water channel; 1 - outer and 2 - inner nuclear membranes

Thus, membrane stability depends on the number and size of lipid pores formed at the defect sites of the liquid-crystalline structure of The Lipid Bilayer. Lipid pores arise from thermal fluctuations and can also be formed under any mechanical, physical, or chemical influences that act as risk factors for membrane stabilization. In such cases, membrane stability will depend on whether or not the lipid pore exceeds its critical size. In the former case, the membrane will rupture; in the latter, its structure will be preserved.

In all eukaryotes, from Yeast to humans, the nuclear envelope is formed by two membranes: the inner and outer. The inner nuclear membrane contains specific proteins that bind nuclear lamins. This membrane is surrounded by the outer nuclear membrane, which is continuous with The Endoplasmic reticulum. The nuclear envelope is perforated by nuclear pores surrounded by pore complexes. The pore complex (a set of proteins) spans the nuclear envelope, joining the lipid bilayer of both membranes around the circumference of the pore into a single entity (Fig. 4.14, C). The pore diameter is 9 nm (which corresponds to the critical diameter of lipid pores), and its length is 15 nm. Through these pores, small molecules and proteins with a molecular mass of 44-45 kDa freely penetrate into (as well as out of) The Nucleus. Other proteins, as well as Nucleic Acids, cross the nuclear pores via Active Transport.

Gap junctions—clusters of membrane channels that connect the Cytoplasm of adjacent cells in Tissues—have a similar protein nature. The diameter of these pores ranges from 1.2 to 2 nm. Biochemical reconstruction of these pores indicates that they are formed by an oligomer of a single protein (for example, in hepatocytes, its molecular mass is 32 kDa). Each gap junction consists of 12 subunits, six from each plasma membrane.

Ion channels facilitate The transport of ~ 10⁶ ions per second down their electrochemical gradient (which significantly exceeds the transport rate of known carrier proteins). These are primarily Na⁺, K⁺, Ca²⁺, and СI^- ions. Protein channels exhibit ion selectivity, meaning that only ions of a specific type pass through them. This implies that each ion must have the appropriate charge and size. Ion channels also differ from pores in that they are not open continuously. They possess gates (Fig. 4.15) that open briefly and then close. The gates open in response to Changes in the resting membrane potential (voltage-gated channels), mechanical stimuli (mechanosensitive channels), or the binding of specific signaling molecules (chemosensitive or Ligand-gated channels). Signaling molecules include Neurotransmitters and NUCLEOTIDES. Over 50 types of ion channels are already known, responsible for the electrical excitability of nerve and Muscle cells, and the transmission of electrical signals along excitable tissues and cells; they are present in all animal cells and in some cells of plants and microorganisms. The most common are potassium channels, which generate the resting membrane potential, and Na⁺ and Ca²⁺ channels, which are involved in the generation of action potentials.

Bioelectric potentials are highly diverse and differ mainly in two parameters: amplitude and frequency. Thus, the discharge of electric fish, generated by a certain number of cells, reaches 800 V, which is sufficient to kill a small animal. At the same time, Brain biopotentials (Electroencephalogram) are in the microvolt range. In terms of oscillation frequency, biopotentials vary from hours (Skin surface) to a few milliseconds (nerve impulses in vertebrates). Electrical activity, regardless of the ions involved in its generation, is divided into two types:

✵ resting membrane potential (RMP), i.e., the potential difference between the Two Sides of the membrane, which primarily depends on the difference in the concentrations of K⁺ ions;

✵ action potentials (AP) - changes in the RMP during cell excitation, caused by Changes in membrane permeability to sodium and Calcium Ions.

Potassium ions penetrate the resting membrane most easily. They leave the cell down their concentration gradient. At the same time, anions remain inside the cell, which gives rise to an electric field—the membrane potential, which tends to pull potassium ions back into the cell. The efflux of potassium ions will stop as soon as the membrane potential (the generation of which is a consequence of potassium efflux) reaches a value at which the electrical driving force "pulling" potassium ions in equals The Effect of the potassium concentration gradient pushing these ions out of the cell, i.e., when the Electrochemical Potential μ = 0. The state of the membrane is then characterized by the absence of transmembrane electric current, equilibrium is reached, and the potential across the membrane is called the equilibrium potassium potential. This potential is determined by the Nernst equation:

where Vk is the equilibrium potassium potential ("minus" inside the cell), Co and Ci are the outer and inner ion concentrations; R is the universal gas constant (2 cal · mol^-1, K); T is the absolute Temperature, K; F is the Faraday constant (2.3 · 10⁴ cal · V^-1 · mol^-1) and Z is the ion charge.

Since other ions also penetrate the membrane at rest (although their permeability is much lower), the RMP value will be lower than the potassium equilibrium potential. For example, the calculated Vk for a squid axon is -90 mV, while the experimentally measured value is -60 mV. This is a result of the influx of primarily sodium ions in the direction opposite to the efflux of potassium ions.

In addition to passive ion transport, the generation of a potential difference across the membrane is also associated with their active transport mechanism (Fig. 4.12). Since the sodium-potassium pump is electrogenic, blocking the Na⁺, K⁺-ATPase will lead to a decrease in the RMP. Importantly, despite The Diversity of living organisms, There are two electrogenic transport ATPases: Na⁺, K⁺-ATPase in animals, and H⁺-ATPase in plants and Fungi. The constant activity of these membrane Enzymes ensures continuous recharging of the membrane, creating an active component of the RMP with a "minus" sign inside.

The mechanisms of excitation were first studied in the giant squid axon: these axons are unmyelinated and have a diameter of up to 1 mm, which facilitates experimentation. Upon excitation of the nerve fiber, the permeability of the plasma membrane to Na⁺ ions increases (the influx is directed into the cell, as their concentration is higher outside), causing a decrease in the RMP—membrane depolarization (Fig. 4.15). This generates the rising phase of the Action Potential. The process of membrane depolarization by sodium ions continues until a new equilibrium state is established, after which the membrane permeability to K⁺ increases sharply. Since potassium ions are more abundant inside the cell, they flow out, repolarizing the membrane back to the initial RMP value.

Fig. 4.15. Diagram of AP GENERATION AND PROPAGATION in a nerve fiber (A-C); AP arising upon stimulation of a plant leaf and propagating through vascular bundles to the roots (D, E): A - AP of a nerve fiber, B - Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF an ion channel (F - selectivity filter, G - gate, S - voltage sensor); C - AP propagation along an excitable membrane: "-" - excited, "+" - unexcited areas of the membrane; arrows indicate the direction of local currents;

D - activation (by light stimulation of leaves) of ³²P uptake by plant roots, E - plant AP

The time-dependent change in membrane permeability during excitation is explained by the specific functioning of sodium and potassium channels (Fig. 4.15, B), which have a selectivity "filter" and a "gate." The selectivity filter "selects" ions based on their physicochemical properties. The gate is a part of the channel whose conformational state depends on the membrane potential. Gates can adopt several alternative Conformations, the stability of which depends on the strength of the electric field. Initial membrane depolarization opens the gates of sodium channels, generating an inward sodium current (curve a, Fig. 4.15, A). Further depolarization not only closes the sodium channel gate but also opens potassium channels, leading to an outward potassium current and, consequently, to membrane repolarization (descending curve b in Fig. 4.15, A). Ion channels acquired this property through evolution. Upon excitation, local currents flow between the unexcited (two) and excited (one) Regions of the membrane (Fig. 4.15, C), which:

✵ repolarize the excited region, which corresponds to the falling phase of the AP and an increase in membrane potassium conductance (Fig. 4.15, A);

✵ depolarize the membrane ahead of the excited region (which is currently generating an AP), and when depolarization reaches the threshold level, an AP is generated. This corresponds to the rising phase of the AP (Fig. 4.15, A) and ensures its unidirectional propagation;

✵ depolarization of the region behind the AP does not excite the membrane because the ion channels, primarily the sodium channel, have not yet had time to recover: the sodium channel is inactivated, which is the refractory state.

The Mechanism of AP generation is the same for all organisms, although there are significant differences. Thus, the depolarizing ion in nerve fibers is Na⁺, in smooth muscle cells it is Ca²⁺, and in plant cells it is Cl-.

The Role of bioelectric potentials in life processes is highly universal and diverse. First, there is the energetic role. The RMP, created by active and passive mechanisms, can be viewed, by analogy with ATP, as a unique form of energy storage in the cell; that is, the criterion of cell "energization" is not only The amount of high-energy compounds in it, but also the magnitude of its membrane potential. It is particularly important that the electrical energy of the membrane is "highly convertible," meaning it can easily transform into Other forms of energy required for specific biological processes. This is especially true for membrane transport, which provides substrates for METABOLISM.

Second, bioelectric potentials play a regulatory role. The electric field strength (10⁵ V/cm) strongly affects individual membrane components—proteins and lipids, whose molecules possess electrical charges and dipole moments. Under The Influence of an electric field, such molecules change their orientation and conformational state. Therefore, changes in the RMP and AP under the action of various factors affect the functioning of membrane enzymes, receptors, channels, pores, transporters, etc.

Third, the informational role of bioelectric processes is essential and is most highly developed in nerve fibers. APs arising in them under the influence of various stimuli propagate at a speed of 100 m/s and ensure the transmission of information from one part of the Organism to another. Similar processes occur in plants as well.

Fourth, bioelectric potentials are of great importance for the self-Organization of living systems. The electric fields around Cells and Tissues act as a force matrix according to which the GROWTH AND DEVELOPMENT of individual Organs of the body take place.