where Ca2+-mediated vesicle fusion occurs. Historically, botulinum toxin was of interest
because of its role in botulism food poisoning. However, botulinum toxin (“botox”) is
now used as a “therapeutic.” Disabling and possibly painful muscle contractions asso-
ciated with neurologic disorders, such as dystonia, can be relieved by injecting the
affected muscle with botulinum toxin. Botox injections are now also used cosmetically.
Injections of the toxin into the muscles over the forehead or around the eyes will
remove “age creases and skin wrinkles,” “restoring the youthful appearance” much
prized in our image-conscious society.
Acetylcholine is also found in non-neuronal tissues. The mammalian respiratory tract
is regulated by ACh; this neurotransmitter also has a direct effect on intestinal smooth
muscle and on the heart. It is therefore reasonable to consider ACh a hormone as much
as a neurotransmitter, as mentioned previously.
4.2.2 The Nicotinic Acetylcholine Receptor4.2.2.1 Isolation of Acetylcholine Receptors
The isolation of the nicotinic acetylcholine receptor glycoprotein was achieved
almost simultaneously in several laboratories (those of Changeux, O’Brien, Brady, and
Eldefrawi) and was helped tremendously by the discovery that the electric organ (elec-
troplax) of the electric eel (Electrophorus electricus, an inhabitant of the Amazon
River) and related species, as well as the electroplax of the electric ray (Torpedo mar-
morata) of the Atlantic Ocean and the Mediterranean Sea, contains acetylcholine
receptors (AChR) in a much higher concentration than, for instance, in human neuro-
muscular endplates or brain tissue.
The discovery that the toxins of Elapidsnakes bind almost irreversibly to the AChR
also facilitated the isolation and study of this receptor. The structure of these venoms
has been elucidated; those most widely used experimentally are the α-bungarotoxin
(BTX) of the Indian cobra and the toxin of the Siamese cobra. These compounds are
peptides containing from 61 to 74 amino acids, five disulfide bridges, and a high pro-
portion of basic arginine and lysine residues, often in close proximity. Venoms are toxic
because they block cholinergic neurotransmission by binding to the receptor.
The AChR is an integral membrane protein, deeply embedded into the postsynaptic
membrane. It can be solubilized by nonionic detergents such as Triton X-100, Tween
80, and others, or anionic detergents such as deoxycholate, a bile acid derivative.
Functionally, the regulation of ion permeability is lost when the receptor is removed
from the membrane; however, the ACh and BTX binding capacity is retained and
can be used for following the course of purification. In a typical isolation procedure,
the electric organ is homogenized in 1 M NaCl with Na 2 HPO 4 and EDTA, which sol-
ubilizes the acetylcholinesterase. The suspension is then centrifuged and the resulting
pellet extracted with detergent, solubilizing the AChR. This receptor solution can
then be purified further by polyacrylamide gel electrophoresis, by affinity partitioning,
or, most efficiently, by affinity chromatography either on an immobilized quaternary
ligand or on Siamese cobra toxin bound to an agarose bead matrix. The specific activ-
ities of the purified preparations range from 8 to 12 μmol of binding sites per gram
of protein, and about 100–150 mg of receptor protein can be obtained from 1 kg of
NEUROTRANSMITTERS AND THEIR RECEPTORS 207