BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 37. EXCITABLE MEMBRANES AND SENSORY SYSTEMS

37.2. Tetrodotoxin and Saxitoxin Block Sodium Channels in Axon Membranes of Nerve Cells

Tetrodotoxin, a potent toxin found in pufferfish, blocks the propagation of nerve impulses along axons and within the excitable membranes of nerve fibers, resulting in respiratory paralysis. The lethal dose for a mouse is approximately 0.01 µg. Due to its high Specificity, tetrodotoxin is widely used in experimental research. It binds very tightly (K ≈ 10^-9 M) to the Na+-channel and blocks the inward flow of sodium ions without affecting the K⁺-channel. Saxitoxin, produced by certain marine dinoflagellates, exhibits an identical mode of action. Shellfish that feed on these dinoflagellates, particularly edible bivalves and mussels, also become toxic. For instance, a single small mussel can contain enough saxitoxin to kill 50 people! A common structural feature of both tetrodotoxin and saxitoxin is the presence of a guanidine group (Fig. 37.4). This positively charged group of the toxin interacts with a negatively charged carboxylate ion located at the Mouth of the channel on the extracellular side of the membrane. Essentially, these toxins act as Competitive Inhibitors of Na⁺.

Class="center">Fig. 37.4. Blockers of the Na⁺-channel

Pufferfish, considered a delicacy in Japan

Owing to their high specificity and strong affinity for the Na⁺-channel, tetrodotoxin and saxitoxins have proven to be invaluable analytical tools. For example, measurements of the binding

of radiolabeled tetrodotoxin with high specific radioactivity have been used to determine the density of Na⁺-channels in various excitable membranes. Cytology/practical/64.html">Unmyelinated nerve fibers, which lack the insulating myelin sheath, typically exhibit a low density of Na⁺-channels—on the order of 20 per 1 µm². In the membranes of such axons, Na+-channels are spaced about 2,000 Å apart. In contrast, within Myelinated nerve fibers, at specialized regions known as nodes of Ranvier, the density of Na⁺-channels reaches remarkably high values—on the order of 10⁴ per 1 µm². The nodes of Ranvier, spaced at intervals of 2 mm along the axon, are the only sites where the axonal membrane of a myelinated nerve comes into contact with the extracellular fluid. The membrane segments between the nodes of Ranvier contain very few channels and do not participate in impulse propagation. The Action Potential jumps from one node to the next, allowing impulses to be conducted much faster and more efficiently than in unmyelinated fibers. The presence of 10⁴ channels per 1 µm² at the node of Ranvier means that a significant portion of the membrane surface in this region is occupied by Na⁺-channels.

The specificity of tetrodotoxin binding to Na⁺-channels has also been instrumental in their Isolation and Purification. To achieve this, integral Membrane Proteins of excitable membranes were solubilized using a detergent and subsequently separated on an ion exchanger. The binding of tetrodotoxin to the solubilized Na+-channel made it possible to quantitatively assay the channel throughout the purification process. The Na+-channel isolated in this manner proved to be a protein with a molecular mass of 230 kDa, composed of several distinct types of subunits. The structural complexity of the Na⁺-channel is presumably due, in part, to the fact that it not only Functions as a highly selective membrane pore but also incorporates a voltage-sensing mechanism (voltage sensor). The charged groups of this Structure respond to Changes in membrane potential during an action potential and transmit this information to the pore-forming region of the channel. Indeed, prior to the onset of the sodium current through the membrane, a preliminary gating current can be detected, which arises from the movement of charged groups within the protein.

Fig. 37.5. Electron micrograph of a myelinated axon from the Spinal Cord. The myelin sheath—a wrapper formed by numerous layers of membranes—acts as an insulator. Conduction velocity in myelinated nerves is significantly higher than in unmyelinated NERVES OF THE same diameter

37.3. Acetylcholine Is a Neurotransmitter

Communication between Nerve Cells occurs at specialized junctions called synapses (Fig. 37.6). Nerve impulses are transmitted across the majority of synapses via chemical Neurotransmitters—small, readily diffusible molecules such as acetylcholine or norepinephrine. Acetylcholine also serves as the neurotransmitter at motor end plates (neuromuscular junctions), which are the contact sites between a motor neuron and Skeletal Muscle fiber. The presynaptic membrane of a cholinergic synapse (i.e., a synapse that utilizes acetylcholine as its neurotransmitter) is separated from the postsynaptic membrane by a synaptic cleft approximately 500 Å wide. The presynaptic axon terminal is packed with synaptic vesicles containing acetylcholine. Upon the arrival of a Nerve Impulse at the synapse, Acetylcholine is released into the synaptic cleft. The acetylcholine molecules then diffuse across the cleft and bind to specific receptor molecules on the postsynaptic membrane. This triggers depolarization of the postsynaptic membrane, after which the wave of depolarization propagates along the electrically excitable membrane of the second nerve Cell. Acetylcholine is subsequently hydrolyzed by acetylcholinesterase, allowing the postsynaptic membrane to repolarize. Acetylcholine is synthesized near the presynaptic axon terminal by The transfer of an acetyl group from acetyl-CoA to Choline. The reaction is catalyzed by the enzyme choline acetyltransferase (choline acetylase). A portion of the newly synthesized acetylcholine is packaged into synaptic vesicles, while the remainder stays in the Cytosol. A single cholinergic synaptic vesicle (typically 400 Å in diameter) contains roughly 10,000 molecules of acetylcholine.

Fig. 37.6. Schematic diagram of a cholinergic synapse

The Study of synaptic function has been greatly advanced by the isolation of synaptosomes from neural tissue homogenates. Synaptosomes are pinched-off presynaptic nerve terminals that reseal into closed structures during the isolation Procedure. They consist of vesicles formed from the presynaptic membrane containing Mitochondria, cytosol, and synaptic vesicles.

37.4. Acetylcholine Opens Cation Channels in the Postsynaptic Membrane

The Resting Potential of the postsynaptic membrane (or motor end plate membrane) is approximately -75 mV. The interaction of acetylcholine with specific receptors induces a dramatic change in the permeability of these membranes (Fig. 37.7). Within 0.1 ms, the conductance for both Na⁺ and K⁺ increases markedly, generating a strong inward Na⁺ current and a weaker outward K⁺ current. The influx of Na⁺ into The Cell leads to the depolarization of the postsynaptic membrane and triggers an action potential in the adjacent postsynaptic neuron or muscle fiber. Acetylcholine opens only a single class of cation channels, which exhibit nearly identical permeability to Na⁺ and K⁺. If the Na+ flux induced by acetylcholine exceeds the K⁺ flux, it is simply due to the steeper electrochemical concentration gradient of Na+ relative to K⁺.

Fig. 37.7. Acetylcholine depolarizes the postsynaptic membrane by increasing Na+ and K+ conductance

Two acetylcholine molecules bind to the receptor molecule, inducing conformational changes that open the channel. The process kinetics, which are consistent with experimental data, can be schematically described by the equation

2А + R5 ⇄ A₂R ⇄ A₂R*,

where A is an acetylcholine molecule, R is the closed channel, and R* is the open channel. During the open channel's half-life of just 1 ms, approximately 10⁴ ions pass through it. Prolonged exposure of the receptor to acetylcholine leads to its desensitization: the channel closes, and the response to acetylcholine disappears for an extended period.