BIOELECTRICAL PHENOMENA IN NERVE CELLS.
The membrane that surrounds the cell is a remarkable structure. It is not only semipermeable, allowing some substances to pass through it and excluding others, but its permeability can be varied. It is generally referred to as the plasma membrane. The nucleus is also surrounded by a membrane, and the organelles are surrounded by or made up of membrane.
Although the chemical structure of membranes and their properties vary onsiderably from one location to another, they have certain common features. They are generally about 7,5 nm (75 Angstrom units) thick. They are made up primarily of protein and lipids. The major lipids are phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The head end of the molecule contains the phosphate portion, is positively charged, and is quite soluble in water (polar, hydrophilic). The tails are quite insoluble (nonpolar, hydrophobia). In the membrane, the hydrophilic ends are exposed to the aqueous environment that bathes the exterior of the cells and the aqueous cytoplasm; the hydrophobic ends meet in the water-poor interior of the membrane. However, there is a degree of asymmetry in the distribution of lipid in the membrane; in human red cells, for example, there is more phosphatidylethanolamine and phosphatidylserine in the inner lamella and more lecithin and sphingomyelin in me outer lamella. The significance of this asymmetry is unknown. In prokaryotes (cells like bacteria in which there is no nucleus), phospholipids are generally the only membrane lipids, but in eukaryotes (cells containing nuclei), cell membranes also contain cholesterol (in animals) or other steroids (in plants). The cholesterol/phospholipid ratio in the membrane is inversely proportionate to the fluidity of the membrane. Variations in the ratio lead to abnormalities of cell function. In healthy animals, the ratio is maintained at a nearly constant level by a variety of regulatory mechanisms.
There are many different proteins embedded in the membrane. They exist as separate globular units and stud the inside and outside of the membrane in a random array. Some are located in the inner surface of the membrane; some are located on the outer surface; and some extend through the membrane (through and through proteins). In general, the uncharged, hydrophobic portions of the protein molecules are located in the interior of the membrane and the charged, hydrophilic portions are located on the surfaces. Some of the proteins contain lipids (lipoproteins) and some contain arbohydrates (glycoproteins). There are 5 types of proteins in the membrane. In addition to structural proteins, there are proteins that function as pumps, actively transporting ions across the membrane. Other proteins function as passive channels for ions that can be opened or closed by changes in the conformation of the protein. A fourth group of proteins functions as receptors that bind neurotransmitters and hormones, initiating physiologic changes inside the cell. A fifth group functions as enzymes, catalyzing reactions at the surfaces of the membrane.
The protein structure – and particularly the enzyme content – of biologic membranes varies not only from cell to cell but also within the same cell. For example, there are different enzymes embedded in cell membranes than in mitochondrial membranes; in epithelial cells, the enzymes in the cell membrane on the mucosal surface differ from those in the cell membrane on the lateral margins of the cells. The membranes are dynamic structures, and their constituents are being constantly renewed at different rates. In addition, some proteins move laterally in the membrane. For example, receptors move in the membrane and aggregate at sites of endocytosis. There is evidence that the lateral movement of components in the membrane is not random but is controlled by mtracellular mechanisms that probably involve microfilaments and nucrotubules.
Underlying most cells is a thin, fuzzy layer plus some fibrils that collectively make up the basement membrane or, more properly, the basal lamina. The material that makes up the basal lamina has been shown to be made up of a collagen derivative plus 2 glycoproteins.
The Sodium-Potassium Pump
The sodium-potassium pump responsible for the coupled active transport of Na+ out of cells and K+ into cells is a unique protein in the cell membrane Figure 1. This protein is also an adenosine triphosphatase, ie, an enzyme that catalyzes the hydrolysis of ATP to adenosine diphosphate (ADP), and it is activated by Na+ and K+. Consequently, it is known as sodium-potassium-activated adenosine triphosphatase (Na+-K+ ATPase). The ATP provides the energy for transport. The pump extrudes three Na+ from the cell for each two K+ it takes into the cell, ie, it has a coupling ratio of 3/2. Its activity is inhibited by ouabain and related digitalis glycosides used in the treatment of heart failure. It is made up of two a subunits, each with a molecular weight of about 95,000, and two b subunits, each with a molecular weight of about 40,000. Separation of the subunits leads to loss of ATPase activity.
The a subunits contain binding sites for ATP and ouabain, whereas the b subunits are glycoproteins. Application of ATP by micropipette to the inside of the membrane increases transport, whereas application of ATP to the outside of the membrane has no effect. Conversely, ouabain inhibits transport when applied to the outside but not to the inside of the membrane. Consequently, the a subunits must extend through the cell membrane. The protein could exist in 2 conformational states. In one, three Na+ bind to sites accessible only from the inside of the membrane. This triggers hydrolysis of ATP, and the protein changes its conformation so that the three Na+ are extruded into the ECP. In the second conformation, two K+ bind to sites accessible only from the outside of the membrane. This triggers a return to the original conformation while extruding two K+ into the interior of the cell. It appears that Na+ binding is associated with phosphorylation of the protein and K+ binding with dephosphorylation.
Effects of Na+ & K+ Transport
It should be noted that since Na+-K+ ATPase transports three Na+ out of the cell for each two K+ it transports in, it is an electrogenic pump, ie, it produces net movement of positive charge out of the cell. The amount of Na+ provided to the pump can be the rate-limiting factor in its operation; consequently, the amount of Na+ extruded by the cell is partly regulated in a feedback fashion by the amount of Na+ in the cell.
The enzyme described above is found in all parts of the body. In some tissues, the active transport of Na+ is coupled to the transport of other substances (secondary active transport). For example the membranes of mucosal cells in the small intestine contain a cotransport protein (symport) that transports glucose into the cell only if Na+ binds to the protein and is transported down its electrochemical gradient at the same time. The electrochemical gradient is maintained by the active transport of Na+ out of the mucosal cell into ECF. Other nutrients are reabsorbed in a similar fashion. In the brain transport by Na+-K+ ATPase is coupled to the reuptake of neurotransmitters. In the heart Na+-K+ ATPase indirectly affects Ca2+ transport because an exchange protein (antiport) in the membranes of cardiac muscle cells exchanges intracellular Ca2+ for extracellular Na+ on an electrically neutral 1 for 2 basis. The rate of this exchange is proportionate to the concentration gradient for Na+ across the cell membrane. If the operation of the Na+-K+ ATPase is inhibited (eg, by ouabam), less intracellular Ca2+ is extruded and intracellular Ca2+ is increased. This facilitates the contraction of cardiac muscle and is the probable explanation of the positively inotropic effect of digitalis glycosides.
Active transport of Na+ and K+ is one of the major energy-using processes in the body and probably accounts for a large part of the basal metabolism. Furthermore, there is a direct link between Na+ and K+ transport and metabolism; the greater the rate of pumping, the more ADP is formed and the available supply of ADP determines the rate at which ATP is formed by oxidative phosphorylation.
In animals, the maintenance of normal cell volume and pressure depend on Na+ and K+ pumping In the absence of such pumping, Cl– and Na+ would enter the cells down their concentration gradients, and water would follow along the osmotic gradient thus created, causing the cells to swell until the pressure inside them balanced the influx. This does not occur, and the osmolality of the cells remains the same as that of the interstitial fluid because Na+ and K+ are actively transported. The membrane potential is maintained, and [Cli– ], the Cl– concentration inside the cells, remains low.
Proteins closely related to Na+-K+ ATPase transport Ca2+ out of cells (Ca2+ ATPase) and extrude H+ from cells in exchange for K+ (H+-K+ ATPase). No other extramitochondrial membrane pumps have been described that can convert energy directly into transportation of ions against concentration gradients, and all other known examples of net extrusion or accumulation of substances across an animal cell membrane against a concentration gradient have been attributed to secondary active transport, ie, they depend on an electrochemical gradient created by Na+-K+ ATPase
In certain situations proteins enter cells, and a number of hormones and other substances secreted by cells are proteins or large polypeptides. Proteins and other large molecules enter cells by the process of endocytosis and are secreted by exocytosis. These processes provide an explanation of how large molecules can enter and leave cells without disrupting cell membranes.
CELL MEMBRANE & RESTING MEMBRANE POTENTIALS
Membrane Potentials
There is a potential difference across the membranes of most if not all cells, with the inside of the cells negative to the exterior. By convention, this resting membrane potential (steady potential) is written with a minus sign, signifying that the inside is negative relative to the exterior. Its magnitude vanes considerably from tissue to tissue, ranging from -9 to –100 mV
Cell membranes are practically impermeable to intracellular protein and other organic anions, which make up most of the intracellular anions and are usually represented by the symbol A-. However, they are moderately permeable to Na+ and rather freely permeable to Cl– and K+. K+ permeability is 50-100 times greater than Na+ permeability. Particle size affects the movement of ions across cell membranes, and it should be noted that the ions in the body are hydrated. Thus, although the atomic weight of potassium (39) is greater than the atomic weight of sodium (23), the hydrated sodium ion – ie, Na+ with its full complement of water – is larger than the hydrated potassium ion. However, it is clear that ions cross membranes via ion channels rather than simple pores. These channels are usually passages through protein molecules, and charge configurations around them and related variables make them relatively specific. Thus, for example, there are separate Na+ channels, K+ channels, and Cl– channels. The ease with which ions pass through some of these channels is controlled (gated) by voltage or by agents such as neurotransmitters. Thus, for example, passage of Na+ through the Na+ channels in excitable tissues (nerve and muscle) is greatly increased by a decrease in membrane potential, ie, they are voltage-gated. At synaptic junctions and elsewhere, many ion channels are chemically gated, ie, their ability to pass ions is increased by the binding of a given neurotransmitter or hormone to receptors associated with them.
Latent Period
If the axon is stimulated and a conducted impulse occurs, a characteristic series of potential changes is observed as the impulse passes the exterior electrode. When the stimulus is applied, there is a brief irregular deflection of the baseline, the stimulus artifact. This artifact is due to current leakage from the stimulating electrodes to the recording electrodes. It usually occurs despite careful shielding, but it is of value because it marks on the cathode-ray screen the point at which the stimulus was applied.
The stimulus artifact is followed by an isopotential interval (latent period) that ends with the next potential change and corresponds to the time it takes the impulse to travel along the axon from the site of stimulation to the recording electrodes. Its duration is proportionate to the distance between the stimulating and recording electrodes and the speed of conduction of the axon. If the duration of the latent period and the distance between the electrodes are known, the speed of conduction in the axon can be calculated. For example, assume that the distance between the cathodal stimulating electrode and the exterior electrode is
The first manifestation of the approaching impulse is a beginning depolarization of the membrane. After an initial 15 mV of depolarization, the rate of depolarization increases. The point at which this change in rate occurs is called the firing level. Thereafter, the tracing on the oscilloscope rapidly reaches and overshoots the isopotential (zero potential) line to approximately +35 mV. It then reverses and falls rapidly toward the resting level. When repolarization is about 70 % completed, the rate of repolarization decreases and the tracing approaches the resting level more slowly. The sharp rise and rapid fall are the spike potential of the axon, and the slower fall at the end of the process is the after-depolarization. After reaching the previous resting level, the tracing overshoots slightly in the hyperpolarizing direction to form the small but prolonged after-hyperpolarization. The after-depolarization is sometimes called the negative after-potential and the after-hyperpolarization the positive after-potential, but the terms are now rarely used. The whole sequence of potential changes is called the action potential. It is a monophasic action potential because it is primarily in one direction. Before electrodes could be inserted in the axons, the response was approximated by recording between an electrode on intact membrane and an electrode on an area of nerve that had been damaged by crushing, destroying the integrity of the membrane. The potential difference between an intact area and such a damaged area is called a demarcation potential.
The proportions of the tracing are intentionally distorted to illustrate the various components of the action potential. Note that the rise of the action potential is so rapid that it fails to show clearly the change in depolarization rate at the firing level, and also that the after-hyperpolarization is only about 1-2 mV in amplitude although it lasts about 40 ms. The duration of the afterdepolarization is about 4 ms in this instance. It is shorter and less prominent in many other neurons. Changes may occur in the after-polarization without changes in the rest of the action potential. For example, if the nerve has been conducting repetitively for a long time, the after-hyperpolarization is usually quite large.
Conduction in myelinated axons depends upon a similar pattern of circular current flow. However, myelin is an effective insulator, and current flow through it is negligible. Instead, depolarization in myelinated axons jumps from one node of Ranvier to the next, with the current sink at the active node serving to electrotonically depolarize to the firing level the node ahead of the action potential. This jumping of depolarization from node to node is called saltatory conduction. It is a rapid process, and myelinated axons conduct up to 50 times faster than the fastest unmyelinated fibers. Indeed, myelination saves considerable space in the nervous system, because in unmyelinated neurons conduction velocity increases with the square root of the diameter of the axon, whereas in myelinated axons the conduction velocity increases directly with the diameter of the axon. Thus, a nervous system made up of unmyelinated axons would have to be many times larger.
Resting membrane potential
a) Common characteristic (There is a potential difference across the membranes of most if not all cells, with the inside of the cells negative to the exterior. By convention, this resting membrane potential (steady potential) is written with a minus sign, signifying that the inside is negative relative to the exterior. Its magnitude vanes considerably from tissue to tissue, ranging from -9 to –100 mV. When 2 electrodes are connected through a suitable amplifier to a CRO and placed on the surface of a single axon, no potential difference is observed. However, if one electrode is inserted into the interior of the cell, a constant potential difference is observed, with the inside negative relative to the outside of the cell at rest. This resting membrane potential is found in almost all cells. In neurons, it is usually about –70 mV.)
b) Mechanism of development (There are two kind of ion’s transport: active and passive. Active transport is doing due to the energy of ATP. The sodium-potassium pump responsible for the coupled active transport of Na+ out of cells and K+ into cells is a unique protein in the cell membrane. This protein is also an adenosine triphosphatase, ie, an enzyme that catalyzes the hydrolysis of ATP to adenosine diphosphate (ADP), and it is activated by Na+ and K+. Consequently, it is known as sodium-potassium-activated adenosine triphosphatase (Na+-K+ ATPase). The ATP provides the energy for transport. The pump extrudes three Na+ from the cell for each two K+ it takes into the cell, ie, it has a coupling ratio of 3/2. Its activity is inhibited by ouabain and related digitalis glycosides used in the treatment of heart failure. It is made up of two a subunits, each with a molecular weight of about 95,000, and two b subunits, each with a molecular weight of about 40,000. Separation of the subunits leads to loss of ATPase activity. The a subunits contain binding sites for ATP and ouabain, whereas the b subunits are glycoproteins. Application of ATP by micropipette to the inside of the membrane increases transport, whereas application of ATP to the outside of the membrane has no effect. Conversely, ouabain inhibits transport when applied to the outside but not to the inside of the membrane. Consequently, the a subunits must extend through the cell membrane. The protein could exist in 2 conformational states. In one, three Na+ bind to sites accessible only from the inside of the membrane. This triggers hydrolysis of ATP, and the protein changes its conformation so that the three Na+ are extruded into the ECP. In the second conformation, two K+ bind to sites accessible only from the outside of the membrane. This triggers a return to the original conformation while extruding two K+ into the interior of the cell. It appears that Na+ binding is associated with phosphorylation of the protein and K+ binding with dephosphorylation.)
Ionic Basis of Resting Membrane Potential
In nerves, as in other tissues, Na+ is actively transported out of the cell and K+ is actively transported in. K+ diffuses back out of the cell down its concentration gradient, and Na+ diffuses back in, but since the permeability of the membrane to K+ is much greater than it is to Na+ at rest, the passive K+ efflux is much greater than the passive Na+ influx. Since the membrane is impermeable to most of the anions in the cell, the K+ efflux is not accompanied by an equal flux of anions and the membrane is maintained in a polarized state, with the outside positive relative to the inside.
In nerve, as in other tissues, a slight decrease in resting membrane potential leads to increased movement of K+ out of and C1- into the cell, restoring the resting membrane potential. Ierve and muscle, however, there is a unique change in the cell membrane when depolarization exceeds 7 mV. This change is a voltage-dependent increase in membrane permeability to Na+, so that the closer the membrane potential is to the firing level the greater the Na+ permeability. The electrical and concentration gradients for Na+ are both directed inward. During the local response, Na+ permeability is slightly increased, but K+ efflux is able to restore the potential to the resting value. When the firing level is reached, permeability is great enough so that Na+ influx further lowers the membrane potential and Na+ permeability is further increased. The consequent Na+ influx swamps the repolarizing processes, and runaway depolarization results, producing the spike potential.
The equilibrium potential for Na+ in mammaliaeurons, calculated by using the Nemst equation, is about +60 mV. With the great increase in Na+ permeability at the start of the action potential, the membrane potential approaches this value. It does not reach it, however, primarily because the change in Na+ permeability is short-lived. Na+ permeability starts to return to the resting value during the rising phase of the spike potential, and Na+ conductance is decreased during repolarization. In addition, the direction of the electrical gradient for Na+ is reversed during the overshoot because the membrane potential is reversed. These factors limit Na+ influx and help bring about repolarization.
Another important factor producing repolarization of the nerve membrane is the increase in K+ permeability that follows the increase in Na+ permeability. The change in K+ permeability starts more slowly and reaches a peak during the falling phase of the action potential. The increase in permeability decreases the barrier to K+ diffusion, and K+ consequently leaves the cell. The resulting net transfer of positive charge out of the cell completes repolarization.
The changes in membrane permeability during the action potential have been documented in a number of ways, perhaps most clearly by the voltage clamp technique. This research technique, the details of which are beyond the scope of this book, has made it possible to measure changes in the conductance of then membrane for various ions. The conductance of an ion is the reciprocal of its electrical resistance in a membrane and is a measure of membrane permeability to that ion. There is no change in C1- conductance. Decreasing the external Na+ concentration decreases the size of the action potential but has little effect on the resting membrane potential. The lack of much effect on the resting membrane potential would be predicted from the Goldman equation, since the permeability of the membrane to Na+ at rest is relatively low. Conversely, increasing the external K+ concentration decreases the resting membrane potential.
The Na+ and K+ channels in the axon are separate, and both are voltage-gated. The Na+ channel is a protein with a molecular weight of about 275,000 and a pore diameter of about 0,5 nm. There are many charged groups on the surface of the channel, and these groups shift when the channel moves from the closed to the open position. Opening of the channel is also known as sodium channel activation. The voltage differences across the membrane that cause the channel to open also drive it more slowly into a special closed state called the inactivated state. The channel remains in the inactivated state for a few milliseconds before returning to the resting state. Na+ channels can be blocked by a poison called tetrodotoxin (TTX) without affecting the K+ channels. Conversely, the K+ channels can be blocked by tetraethylammonium (TEA) without any changes in Na+ conductance.
Although Na+ enters the nerve cell and K+ leaves it during the action potential, the number of ions involved is not large relative to the total numbers present. The fact that the nerve gains Na+ and loses K+ during activity has been demonstrated experimentally, but significant differences in ion concentrations can be measured only after prolonged, repeated stimulation.
As noted above, the after-depolarization and the after-hyperpolarization represent restorative processes in the cell that are separate from those causing the action potential. Relatively little is known about their origin. The after-depolarization is reduced by agents that inhibit metabolism. The after-hyperpolarization is also reduced, and it now seems clear that it is due to the electrogenic action of the sodium pump. The net flux of Na+ to the exterior hyperpolarizes the membrane until equilibrium conditions are restored.
A decrease in extracellular Ca2+ increases the excitability of nerve and muscle cells by decreasing the amount of depolarizatioecessary to initiate the changes in the Na+ and K+ conductance that produce the action potential. Conversely, an increase in extracellular Ca2+ “stabilizes the membrane” by decreasing excitability. The concentration and electrical gradients for Ca2+ are directed inward, and Ca2+ enters neurons during the action potential. The early phase of Ca2+ entry is blocked by TTX, and it appears that even though Na+ permeability is much greater than Ca2+ permeability, Ca2+ is entering via the Na+ channels. An additional late phase of Ca2+ entry is unaffected by TTX and TEA and apparently occurs via a separate voltage-sensitive Ca2+ pathway. Ca2+ entering during the delayed phase plays an important role in the secretion of synaptic transmitters, a Ca2+-dependent process. In addition, Ca2+ entry contributes to depolarization, and in some instances in invertebrates it is primarily responsible for the action potential.
The central nervous system contains more than 100 billioeurons. Figure 1 shows a typical neuron of a type found in the brain motor cortex. The incoming signals enter the neuron through synapses mainly on the neuronal dendrites, but also on the cell body. For different types of neurons, there may be only a few hundred or as many as 200,000 such synaptic connections from the input fibers. Conversely, the output signal travels by way of a single axon leaving the neuron, but this axon has many separate branches to other parts of the nervous system or peripheral body.
A special feature of most synapses is that the signal normally passes only in the forward direction (from axon to dendrites). This allows the signal to be conducted in the required directions for performing necessary nervous functions. We shall also see that the neurons are arranged into a multitude of differently organized neural networks that determine the functions of the nervous system.
