BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 37. EXCITABLE MEMBRANES AND SENSORY SYSTEMS

37.5. Acetylcholine Is Released in Quanta

Studies of Nerve Impulse transmission at neuromuscular junctions by Bernard Katz revealed that acetylcholine is released from the presynaptic membrane in packets containing about 10⁴ molecules each. Evidence for the quantal release of acetylcholine came from analyses of the Membrane Potential of motor end plates, which exhibit spontaneous electrical activity even in the absence of nerve stimulation. Depolarizing pulses with an amplitude of 0.5 mV and a duration of about 20 ms occur in bursts. These so-called miniature end-plate potentials arise randomly with a probability that remains constant over long periods. A miniature end-plate potential is elicited by the spontaneous release of a single synaptic vesicle. The full end-plate depolarization produced by an Action Potential results from the synchronous release of roughly 100 "quanta" ("packets") of acetylcholine in less than 1 ms. The number of released acetylcholine quanta depends on the potential of the presynaptic membrane. In other words, acetylcholine release represents an electrically regulated form of secretion. Furthermore, the release of acetylcholine requires the presence of Ca²⁺ in the extracellular fluid. Depolarization of the presynaptic membrane triggers an influx of Ca²⁺,

which promotes the transient fusion of synaptic vesicles with the presynaptic membrane.

37.6. Reconstituted Membrane Vesicles Become Cation-Permeable upon the Addition of Acetylcholine

Recent years have witnessed major advances in the purification of acetylcholine receptors and the reconstitution of functionally active membrane vesicles. The most suitable Starting Material for such research is the electric organ of electric fish, such as *Torpedo* (an electric ray), which is exceptionally rich in cholinergic postsynaptic membranes. The electric organ is composed of columns of Cells called electroplaques. One side of these cells (the innervated face) bears nerve endings and is electrically excitable, whereas the opposite side (the noninnervated face) is heavily folded and electrically inexcitable. The potential difference generated upon stimulation of the electroplaques results from the response Asymmetry of the two surfaces. In an electric fish such as *Electrophorus*, the membrane potential of the innervated face shifts from -90 to +60 mV upon excitation, whereas the noninnervated face maintains a potential of -90 mV. Consequently, at the peak of the action potential, the potential difference between the outer surfaces of the two sides is 150 mV (Fig. 37.8). The electroplaques of the electric organ are arranged in parallel, so their potential differences add up. An organ consisting of 5000 rows of electroplaques can thus generate a discharge of 750 V. Interestingly, the electroplaques of the electric eel evolved from Muscle cells, retaining the electrically excitable outer muscle membrane while losing the contractile apparatus. The electric organ of *Electrophorus* serves as an outstanding source of Na⁺ channels.

Class="center">*Electrophorus electricus*, the American electric eel

Fig. 37.8. Voltage generation in an electroplaque of the electric eel

Another exotic biological material has proven to be an invaluable source of acetylcholine receptors. To identify the receptor within a mixture of macromolecules, it must be specifically labeled. Snake neurotoxins are used for this purpose, specifically α-bungarotoxin from the venom of a Taiwanese snake and cobratoxin (from cobra venom). These neurotoxins block neuromuscular transmission by binding to acetylcholine receptors on motor end

plates or on the innervated surface of electroplaques in the electric organ. Neurotoxins are small basic Proteins (7 kDa) that can be radioactively labeled to a high specific activity either by iodination with iodine-125 or by conversion into a Schiff base with Pyridoxal phosphate followed by reduction of the resulting product with ³H-borohydride. Labeled cobratoxin binds tightly to the Acetylcholine Receptor (with a dissociation constant on the order of 10^-9 M) and, crucially, exhibits virtually no binding to other macromolecules of the postsynaptic membrane. Thus, the acetylcholine receptor can be specifically tagged with a radioactive atom.

Fig. 37.9. Three-dimensional Structure of a neurotoxin that blocks the acetylcholine receptor. This neurotoxin is produced by sea snakes

By treating membrane fragments with a nonionic detergent (such as the polyoxyethylene derivative Tween-80), researchers succeeded in solubilizing acetylcholine receptors from the electric organ. The resulting solution was fractionated using Gel filtration and Ion-exchange chromatography. The final purification step involved Affinity Chromatography on a Column containing covalently bound cobratoxin, yielding a 10,000-fold purified receptor. The acetylcholine receptor is a 270-kDa complex composed of four types of subunits. The 40-kDa subunit is affinity-labeled by radioactive compounds containing a trimethylammonium group, indicating that it houses the acetylcholine-binding site. Membrane vesicles containing purified acetylcholine receptors were prepared by adding Phospholipids to the receptor solution and subsequently removing the detergent via dialysis. It was demonstrated that radioactive sodium ions (²²Na⁺), incorporated into the internal aqueous space of the vesicles during reconstitution, are released upon The addition of acetylcholine or its analogs, such as carbamoylcholine (Fig. 37.10). This sodium ion release is blocked by bungarotoxin and conventional acetylcholine antagonists, proving that it is mediated by the specific interaction of acetylcholine with the membrane-bound receptor.

Fig. 37.10. Acetylcholine triggers the release of Na⁺ from reconstituted membrane vesicles containing the acetylcholine receptor. The ordinate represents the ²²Na⁺ content in the synaptic vesicles

37.7. Acetylcholine Is Rapidly Hydrolyzed, Repolarizing the End Plate

Restoring the excitability of the postsynaptic membrane requires turning off the depolarizing signal, a function carried out by acetylcholinesterase, discovered by David Nachmansohn in 1938. The enzyme hydrolyzes acetylcholine into acetate and Choline, returning the permeability of the postsynaptic membrane to its resting level and repolarizing it. Acetylcholinesterase is localized within the synaptic cleft, where it is anchored to a matrix of Collagen and glycosaminoglycans derived from the postsynaptic Cell. The enzyme has a mass of 260 kDa and an α₂β₂ subunit structure. Acetylcholinesterase can be readily dissociated from acetylcholine receptors. It is characterized by a remarkably high turnover number of 25,000 s^-1, meaning a single molecule of acetylcholine is cleaved in just 40 µs. This extraordinarily high catalytic rate is crucial for the rapid restoration of the polarized state of the postsynaptic membrane. Synapses are able to transmit a thousand impulses per second precisely because the postsynaptic membrane repolarizes in fractions of a millisecond.

Fig. 37.11. Electron micrograph of acetylcholine receptors in the postsynaptic membrane

In terms of its MECHANISM OF ACTION, acetylcholinesterase is similar to Chymotrypsin. Acetylcholine interacts with a specific Serine residue at the Active Site of acetylcholinesterase, forming a covalently bound acetyl-enzyme intermediate, while choline is released. The acetyl-enzyme then reacts with a Water molecule, yielding acetate and the regenerated free enzyme (Fig. 37.12).

Fig. 37.12. Mechanism of the catalytic action of acetylcholinesterase