Apamin is an 18 amino acid globular

peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...

neurotoxin Neurotoxins are toxins that are destructive to nerve tissue (causing neurotoxicity). Neurotoxins are an extensive class of exogenous chemical neurological insultsSpencer 2000 that can adversely affect function in both developing and matur ...

found in

apitoxin Apitoxin or bee venom is the venom produced by the honey bee. It is a cytotoxic and hemotoxic bitter colorless liquid containing proteins, which may produce local inflammation. It may have similarities to sea nettle toxin. Components Bee venom i ...

(

bee Bees are winged insects closely related to wasps and ants, known for their roles in pollination and, in the case of the best-known bee species, the western honey bee, for producing honey. Bees are a monophyly, monophyletic lineage within the ...

venom Venom or zootoxin is a type of toxin produced by an animal that is actively delivered through a wound by means of a bite, sting, or similar action. The toxin is delivered through a specially evolved ''venom apparatus'', such as fangs or a ...

). Dry bee venom consists of 2–3% of apamin. Apamin selectively blocks

SK channel SK channels (small conductance calcium-activated potassium channels) are a subfamily of calcium-activated potassium channels. They are so called because of their small single channel conductance in the order of 10 pS. SK channels are a type of i ...

s, a type of Ca²⁺-activated K⁺ channel expressed in the

central nervous system The central nervous system (CNS) is the part of the nervous system consisting primarily of the brain and spinal cord. The CNS is so named because the brain integrates the received information and coordinates and influences the activity of all p ...

. Toxicity is caused by only a few amino acids, in particular cysteine₁, lysine₄, arginine₁₃, arginine₁₄ and histidine₁₈. These amino acids are involved in the binding of apamin to the Ca²⁺-activated K⁺ channel. Due to its specificity for SK channels, apamin is used as a drug in biomedical research to study the electrical properties of SK channels and their role in the

afterhyperpolarization Afterhyperpolarization, or AHP, is the hyperpolarizing phase of a neuron's action potential where the cell's membrane potential falls below the normal resting potential. This is also commonly referred to as an action potential's undershoot pha ...

s occurring immediately following an

action potential An action potential occurs when the membrane potential of a specific cell location rapidly rises and falls. This depolarization then causes adjacent locations to similarly depolarize. Action potentials occur in several types of animal cells, ...

Origin

The first symptoms of apitoxin (bee venom), that are now thought to be caused by apamin, were described back in 1936 by Hahn and Leditschke. Apamin was first isolated by Habermann in 1965 from ''Apis mellifera'', the

Western honey bee The western honey bee or European honey bee (''Apis mellifera'') is the most common of the 7–12 species of honey bees worldwide. The genus name ''Apis'' is Latin for "bee", and ''mellifera'' is the Latin for "honey-bearing" or "honey carrying", ...

. Apamin was named after this bee. Bee

contains many other compounds, like histamine, phospholipase A2, hyaluronidase, MCD peptide, and the main active component

melittin Melittin is the main component (40–60% of the dry weight) and the major pain producing substance of honeybee (''Apis mellifera'') venom. Melittin is a basic peptide consisting of 26 amino acids. Function The principal function of melittin ...

. Apamin was separated from the other compounds by gel filtration and ion exchange chromatography.

Structure and active site

Apamin is a

polypeptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides ...

possessing an

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...

sequence of H-Cys-Asn-Cys-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Cys-Ala-Arg-Arg-Cys-Gln-Gln-His-NH₂ (one-letter sequence CNCKAPETALCARRCQQH-NH₂, with disulfide bonds between Cys₁-Cys₁₁ and Cys₃-Cys₁₅). Apamin is very rigid because of the two disulfide bridges and seven hydrogen bonds. The three-dimensional structure of apamin has been studied with several spectroscopical techniques: HNMR, Circular Dichroism,

Raman spectroscopy Raman spectroscopy () (named after Indian physicist C. V. Raman) is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman s ...

, FT-IR. The structure is presumed to consist of an alpha-helix and beta-turns, but the exact structure is still unknown. By local alterations it is possible to find the amino acids that are involved in toxicity of apamin. It was found by Vincent et al. that guanidination of the ε-amino group of lysine₄ does not decrease toxicity. When the ε-amino group of lysine₄ and the α-amino group of cysteine₁ are acetylated or treated with fluorescamine, toxicity decreases with a factor of respectively 2.5 and 2.8. This is only a small decrease, which indicates that neither the ε-amino group of lysine₄ nor the α-amino group of cysteine₁ is essential for the toxicity of apamin. Glutamine₇ was altered by formation of an amide bond with glycine ethyl ester, this resulted in a decrease in toxicity of a factor 2.0. Glutamine₇ also doesn't appear to be essential for toxicity. When histidine₁₈ is altered by carbethoxylation, toxicity decreases only by a factor 2.6. But when histidine₁₈, the ε-amino group of lysine₄ and the α-amino group of cysteine₁ all are carbethoxylated and acetylated toxicity decreases drastically. This means that these three amino acids are not essential for toxicity on their own, but the three of them combined are. Chemical alteration of arginine₁₃ and arginine₁₄ by treatment of

1,2-cyclohexanedione 1,2-Cyclohexanedione is an organic compound with the formula (CH)(CO). It is one of three isomeric cyclohexanediones. It is a colorless compound that is soluble in a variety of organic solvents. It can be prepared by oxidation of cyclohexanone ...

and cleavage by

trypsin Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the d ...

decreases toxicity by a factor greater than 10. The amino acids that cause toxicity of apamin are cysteine₁, lysine₄, arginine₁₃, arginine₁₄ and histidine₁₈.

Toxicodynamics

Apamin is the smallest neurotoxin polypeptide known, and the only one that passes the blood-brain barrier. Apamin thus reaches its target organ, the central nervous system. Here it inhibits small-conductance Ca²⁺-activated K⁺ channels (SK channels) in neurons. These channels are responsible for the afterhyperpolarizations that follow action potentials, and therefore regulate the repetitive firing frequency. Three different types of SK channels show different characteristics. Only SK2 and SK3 are blocked by apamin, whereas SK1 is apamin insensitive. SK channels function as a tetramer of subunits. Heteromers have intermediate sensitivity. SK channels are activated by the binding of intracellular Ca²⁺ to the protein

calmodulin Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the bi ...

, which is constitutively associated to the channel. Transport of potassium ions out of the cell along their concentration gradient causes the membrane potential to become more negative. The SK channels are present in a wide range of excitable and non-excitable cells, including cells in the central nervous system, intestinal myocytes, endothelial cells, and hepatocytes. Binding of apamin to SK channels is mediated by amino acids in the pore region as well as extracellular amino acids of the SK channel. It is likely that the inhibition of SK channels is caused by blocking of the pore region, which hinders the transport of potassium ions. This will increase the neuronal excitability and lower the threshold for generating an

. Other toxins that block SK channels are tamapin and scyllatoxin.

Toxicokinetics

The kinetics of labeled derivatives of apamin were studied in vitro and in vivo in mice by Cheng-Raude et al. This shed some light on the kinetics of apamin itself. The key organ for excretion is likely to be the

kidney The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; bloo ...

, since enrichment of the labeled derivatives was found there. The peptide apamin is small enough to pass the glomerular barrier, facilitating renal excretion. The central nervous system, contrarily, was found to contain only very small amounts of apamin. This is unexpected, as this is the target organ for neurotoxicity caused by apamin. This low concentration thus appeared to be sufficient to cause the toxic effects. However, these results disagree with a study of Vincent et al. After injection of a supralethal dose of radioactive acetylated apamin in mice, enrichment was found in the

spinal cord The spinal cord is a long, thin, tubular structure made up of nervous tissue, which extends from the medulla oblongata in the brainstem to the lumbar region of the vertebral column (backbone). The backbone encloses the central canal of the spin ...

, which is part of the target organ. Some other organs, including kidney and brain, contained only small amounts of the apamin derivative.

Symptoms

Symptoms following bee sting may include: *local effects: burning or stinging

pain Pain is a distressing feeling often caused by intense or damaging stimuli. The International Association for the Study of Pain defines pain as "an unpleasant sensory and emotional experience associated with, or resembling that associated with, ...

, swelling, redness. *severe systemic reactions: swelling of the

tongue The tongue is a muscular organ in the mouth of a typical tetrapod. It manipulates food for mastication and swallowing as part of the digestive process, and is the primary organ of taste. The tongue's upper surface (dorsum) is covered by taste bu ...

and

throat In vertebrate anatomy, the throat is the front part of the neck, internally positioned in front of the vertebrae. It contains the pharynx and larynx. An important section of it is the epiglottis, separating the esophagus from the trachea (windpi ...

, difficulty

breathing Breathing (or ventilation) is the process of moving air into and from the lungs to facilitate gas exchange with the internal environment, mostly to flush out carbon dioxide and bring in oxygen. All aerobic creatures need oxygen for cell ...

, and shock. *development of

optic neuritis Optic neuritis describes any condition that causes inflammation of the optic nerve; it may be associated with demyelinating diseases, or infectious or inflammatory processes. It is also known as optic papillitis (when the head of the optic nerv ...

and

atrophy Atrophy is the partial or complete wasting away of a part of the body. Causes of atrophy include mutations (which can destroy the gene to build up the organ), poor nourishment, poor circulation, loss of hormonal support, loss of nerve supply ...

. * atrial fibrillation,

cerebral infarction A cerebral infarction is the pathologic process that results in an area of necrotic tissue in the brain (cerebral infarct). It is caused by disrupted blood supply (ischemia) and restricted oxygen supply (hypoxia), most commonly due to thromboemb ...

, acute

myocardial infarction A myocardial infarction (MI), commonly known as a heart attack, occurs when blood flow decreases or stops to the coronary artery of the heart, causing damage to the heart muscle. The most common symptom is chest pain or discomfort which ...

, Fisher's syndrome, acute inflammatory

polyradiculopathy Radiculopathy, also commonly referred to as pinched nerve, refers to a set of conditions in which one or more nerves are affected and do not work properly (a neuropathy). Radiculopathy can result in pain (radicular pain), weakness, altered sensati ...

(

Guillain–Barré syndrome Guillain–Barré syndrome (GBS) is a rapid-onset muscle weakness caused by the immune system damaging the peripheral nervous system. Typically, both sides of the body are involved, and the initial symptoms are changes in sensation or pain often ...

), claw hand (through a central action of apamin on the

and a peripheral action in the form of median and

ulna The ulna (''pl''. ulnae or ulnas) is a long bone found in the forearm that stretches from the elbow to the smallest finger, and when in anatomical position, is found on the medial side of the forearm. That is, the ulna is on the same side of t ...

neuritis Neuritis () is inflammation of a nerve or the general inflammation of the peripheral nervous system. Inflammation, and frequently concomitant demyelination, cause impaired transmission of neural signals and leads to aberrant nerve function. Neuri ...

, causing spasms of the long flexors in the

forearm The forearm is the region of the upper limb between the elbow and the wrist. The term forearm is used in anatomy to distinguish it from the arm, a word which is most often used to describe the entire appendage of the upper limb, but which in ...

). Patients poisoned with bee venom can be treated with

anti-inflammatory Anti-inflammatory is the property of a substance or treatment that reduces inflammation or swelling. Anti-inflammatory drugs, also called anti-inflammatories, make up about half of analgesics. These drugs remedy pain by reducing inflammation as ...

medication,

antihistamine Antihistamines are drugs which treat allergic rhinitis, common cold, influenza, and other allergies. Typically, people take antihistamines as an inexpensive, generic (not patented) drug that can be bought without a prescription and provide ...

s and oral prednisolone. Apamin is an element in

. You can come into contact with apamin through bee venom, so the symptoms that are known are not caused by apamin directly, but by the venom as a whole. Apamin is the only neurotoxin acting purely on the central nervous system. The symptoms of apamin toxicity are not well known, because people are not easily exposed to the toxin alone. Through research about the neurotoxicity of apamin some symptoms were discovered. In mice, the injection of apamin produces convulsions and long-lasting spinal spasticity. Also it is known that the polysynaptic spinal reflexes are disinhibited in cats. Polysynaptic reflex is a reflex action that transfers an impulse from a sensory neuron to a motor neuron via an interneuron in the spinal cord. In rats, apamin was found to cause tremor and ataxia, as well as dramatic haemorrhagic effects in the lungs. Furthermore, apamin has been found to be 1000 times more efficient when applied into the ventricular system instead of the peripheral nervous system. The ventricular system is a set of structures in the brain containing cerebrospinal fluid. The peripheral nervous system contains the nerves and ganglia outside of the brain and spinal cord. This difference in efficiency can easily be explained. Apamin binds to the SK channels, which differ slightly in different tissues. So apamin binding is probably stronger in SK channels in the ventricular system than in other tissues.

Toxicity rates

In earlier years it was thought that apamin was a rather nontoxic compound (LD₅₀ = 15 mg/kg in mice) compared to the other compounds in bee venom. The current lethal dose values of apamin measured in mice are given below. There are no data known specific for humans. Intraperitoneal (mouse) LD₅₀: 3.8 mg/kg Subcutaneous (mouse) LD₅₀: 2.9 mg/kg Intravenous (mouse) LD₅₀: 4 mg/kg Intracerebral (mouse) LD₅₀: 1800 ng/kg Parenteral (mouse) LD₅₀: 600 mg/kg

Therapeutic use

Recent studies have shown that SK channels do not only regulate afterhyperpolarization, they also have an effect on

synaptic plasticity In neuroscience, synaptic plasticity is the ability of synapses to strengthen or weaken over time, in response to increases or decreases in their activity. Since memories are postulated to be represented by vastly interconnected neural circui ...

. This is the activity-dependent adaptation of the strength of synaptic transmission. Synaptic plasticity is an important mechanism underlying learning and memory processes. Apamin is expected to influence these processes by inhibiting SK channels. It has been shown that apamin enhances learning and memory in rats and mice. This may provide a basis for the use of apamin as a treatment for memory disorders and cognitive dysfunction. However, due to the risk of toxic effects, the therapeutic window is very narrow. SK channel blockers may have a therapeutic effect on

Parkinson's disease Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...

. Dopamine, which is depleted in this disease, will be released from midbrain dopaminergic neurons when these SK channels are inhibited. SK channels have also been proposed as targets for the treatment of

epilepsy Epilepsy is a group of non-communicable neurological disorders characterized by recurrent epileptic seizures. Epileptic seizures can vary from brief and nearly undetectable periods to long periods of vigorous shaking due to abnormal electrical ...

, emotional disorders and

schizophrenia Schizophrenia is a mental disorder characterized by continuous or relapsing episodes of psychosis. Major symptoms include hallucinations (typically hearing voices), delusions, and disorganized thinking. Other symptoms include social wi ...

References

External links

* {{MeshName, Apamin Neurochemistry Neurotoxins Ion channel toxins Peptides