Alpha-Latrotoxin

α-Latrotoxin (α-LTX) can naturally be found in widow spiders of the genus Latrodectus. The most famous of those spiders are the black widows, Latrodectus mactans. The venom of widow spiders (Latrodectus) contains several protein toxins, called latrotoxins, which selectively target against either vertebrates, insects or crustaceans. One of these toxins is α-latrotoxin and targets selectively against vertebrates, it is ineffective in insects and crustaceans. α-LTX has a high affinity for receptors that are specific for neuronal and endocrine cells of vertebrates.

Incidents
Bite incidents of Latrodectus occur usually because of accidental contact with the spiders. The species are not aggressive to humans naturally, but may bite as a defence mechanism when threatened. Exposure occurs because the species house in domestic domains, hiding in clothing and furniture. As such, bite incidents occur accidental most of the time.

There are no tests that confirm widow spider bites, or latrodectism symptoms., The diagnosis is clinical and based on historic evidence of widow spider bites. Pathognomonic symptoms such as localized sweating and piloerection provide evidence of envenomation. Furthermore, a doctor must consult the patient of places where exposure to widow spiders can occur.

Species of Latrodectus appear globally, but each continent has their own unique species. A few countries actively research bite incidents of these spiders.

In Australia, extensive research has been done to bite incidents of the red back spider (Latrodectus hasselti): - In 1992, Sutherland reported 258 antivenin administrations in Australia for red back spider bite incidents. Two treatments showed adverse reactions (0.8%). - In 1993, Mead and Jelinek published a report on bite incidents in the period 1979-1988 in a children hospital in Perth. There were 241 bite incidents, mostly in the summer, of which 156 were from red back spiders. Twice as many boys were bitten than girls, with a peak age distribution around the second and third year of age. - In 1991-1992, 32 bite incidents due to red back spiders were reported in Alice Springs Hospital. Of the patients, 26 required antivenin (81%).

In the United States, black widow spiders bites have been reported: - In 1981, Key identified 23 patients over the last 3 years treated with calcium gluconate or methocarbamol for black widow spider envenomation. Treatment of calcium gluconate cured 46% of the patients (total of 13), whereas methocarbanol only relieved 10% of the patients (total of 10). - In 1992, Clark et al. reported that an urban toxicology referral center received 163 patients during a 9 year period (1982–1990). Antivenin was administered to 58 patients (36%), who all had symptomatic relief and were cured mostly within an hour. Of the total patients, 55% of the patients has pain relief using opioid analgesics and with combination of benzodiazepines, 70% had pain relief.

Biosynthesis
As the DNA sequence for α-LTX is transcribed and translated, an inactive precursor molecule of α-LTX (156.9 kDa) is formed. This precursor molecule undergoes post-translational processing where the eventual, active α-LTX protein (131.5 kDa) is formed.

The N-terminus of the α-LTX precursor molecule is preceded by short hydrophilic sequences ending with a cluster of basic amino acids. These clusters are recognized by proteolytic enzymes (furin-like proteases) which cleave and activate the α-LTX precursor molecules by means of hydrolysis. The C-terminus too is recognized by these furin-like proteases and is also cleaved.

α-LTX precursor molecules are synthesized by free ribosomes in the cytosol and are therefore cytosolic in the secretory epithelial cells of the venom glands., They can, however, associate with secretory granules although they are not taken up in the lumen of the granules. The cytosolic α-LTX precursor molecule is released from the cell by means of holocrine secretion where it ends up in the venom gland of the spider. This gland contains the several proteases involved in the cleavage of the precursor α-LTX molecule.

The α-LTX proteins tertiary structure can be divided in three parts: the N-terminal wing (36 kDa), the body (76 kDa) and the C-terminal head (18,5 kDa). Because of C-terminal ankyrin repeats, which mediate protein-protein interactions, the α-LTX monomer forms a dimer with another α-LTX monomer under normal conditions. The body as well as the head contain ankyrin repeats. α-LTX is usually found as a dimer, in which the monomers bind ‘head-to-tail’. Tetramer formation activates toxicity.

Toxicokinetics
Affected cells include motor nerve endings and endocrine cells. No major enzymatic activities are associated with α-LTX. Instead, the toxin can form pores in the lipid membranes and induce Ca2+ ionflow. The onset of effects by intoxication can occur with a lag-period of 1 to 10 minutes, even at subnanomolar concentration levels. At nanomolar concentrations, bursts of neurotransmitter release occur. After the bursts, prolonged periods of steady-state release take effect.,

Stimulation of small end-plate action potentials are initially induced by the neurotoxin, while later on the neurotransmission is blocked at the neuromuscular junction. This is due to depletion of synaptic vesicle contents.

Toxicodynamics
α-LTX in its tetrameric form interacts with receptors (neurexins and latrophilins) on the neuronal membrane, which causes insertion of α-LTX into the membrane.

Once the tetramer is inserted into the cell membrane, two mechanism of actions can occur. First insertion may lead to pore formation and possibly other effects and secondly the receptor may be activated which leads to intracellular signaling. The four heads of the tetramer form a bowl surrounding the pore, which is restricted at one point to 10 Å. Millimolar concentrations of Ca2+ and Mg2+ strongly catalyses tetramer formation, suggesting that the tetrametric state is divalent cation-dependent. while EDTA favours formation of the dimer. Research also shows that concentrations of La3+ higher than 100 µM also block tetramerisation. Pore formation can occur in pure lipid membranes, but reconstituted receptors greatly increase pore formation. Biological membranes block pore formation when no α-LTX receptors are present (neurexin, latrophilin, PTPσ). It is also known that the three highly conserved cysteine residues are involved with α-LTX receptor binding, because mutants containing serine instead of cysteine residues did not induce toxicity. The N-terminal domain needs to fold properly, in which the disulfide bonds need to be functional. The α-LTX toxin is bound by a small protein, LMWP or latrodectin. It has been observed that pore formation in lipid bilayers is impossible when latrodectin is unavailable. Lactrodectin has no effect on α-LTX toxicity.

Pore formation
The pores formed by α-LTX in the membrane are permeable to Ca2+ and therefore allows an influx of Ca2+ into the cell. This influx into an excitable cell stimulates exocytosis directly and efficiently. The cation influx is proportional to the amount of pores and hence the amount of involved receptors expressed on the cell membrane. Also Ca2+ strongly facilitates the forming of the tetramers and so its pore formation. The pore is also permeable to neurotransmitters which causes massive leakage of the neurotransmitter pool in the cytosol.

Alongside the influx of Ca2+, the channel is not very selective, allowing Na+, K+, Ba2+, Sr2+, Mg2+, Li+ and Cs+ to pass the membrane too. The pore is open most of the time, with an open probability of 0.8. Most trivalent cations block channels at 50-100 μM, such as Yb3+, Gd3+, Y3+, La3+ and Al3+.

The pore is not only permeable for cations, but also for water. This causes nerve terminal swelling. Further membrane potential disturbances occur due to permeability of small molecules, such as neurotransmitters and ATP to pass through the α-LTX pore.

Membrane penetration
Although tetrameric pore formation of α-latrotoxin has been shown conclusively, some authors still dispute whether this is the main mode of action of α-latrotoxin, and believe that α-latrotoxin (tetrameric or not) may penetrate through the membrane of target cells to interact directly with intracellular neurotransmitter release machinery.

Receptors
The following mechanism is suggested for receptor-mediated effects. Three receptors for α-latrotoxin have been described:


 * neurexin
 * latrophilin (aka CIRL, Calcium-Independent Receptor for Latrophilin)
 * protein tyrosine phosphatase sigma (PTPσ).

The toxin stimulates a receptor, most likely latrophilin, which is a G-protein coupled receptor linked to Gαq/11. The downstream effector of Gαq/11 is phospholipase C (PLC).When activated PLC increases the cytosolic concentration of IP3, which in turn induces release of Ca2+ from intracellular stores. This rise in cytosolic Ca2+ may increase the probability of release and the rate of spontaneous exocytosis. Latrophilin with α-LTX can induce the activation of Protein Kinase C (PKC). PKC is responsible for the phosphorylation of SNARE proteins. Thus latrophilin with α-LTX induces the effect of exocytosis of transport vesicles. The exact mechanism has to be discovered.

Signaling
As well as the major effects of α-latrotoxin pore formation, other effects of α-latrotoxin are mediated by interaction with latrophilin and intracellular signalling (see signal transduction).

Structure activity relationship (SAR)
The natural occurring α-LTX dimer has to form a tetramer to be toxic. Tetramerisation occurs only in the presence of bivalent cations (such as Ca2+ or Mg2+) or amphipathic molecules. The four monomers that form this tetramer are symmetrically arranged around a central axis, resembling a four-blade propeller with a diameter of 250 Å and a thickness of 100 Å. The head domains form the compact, central mass brought together and surrounded by the body domains. The wings stand perpendicular towards the axis of the tetramer. Because of this form the tetramer contains a pear-shaped channel in the central mass. At the lower end the diameter of this channel is 25Å, then widens to 36 Å to be constricted to 10 Å at the top.

The base of the tetramer (below the wings) is 45Å deep and is hydrophobic which mediates insertion into the cell membrane. Also insertion of the tetramer is only possible in presence of certain receptors (mainly neurexin Iα and latrophilin and PTPσ in a minor extent) on the membrane. Neurexin Iα only mediates insertion under presence of Ca2+, whereas latrophilin and PTPσ can mediate insertion without presence of Ca2+. So because of the channel and the insertion in the cell membrane the protein makes the cell more permeable to substances that can pass through the channel. These substances are mono- and bivalent cations, neurotransmitters, fluoscent dyes and ATP.

Toxicity
The LD50 of α-LTX  in mice is 20-40 µg/kg of body weight. LD50 for frog = 145 mg/kg, blackbird = 5.9, canary = 4.7, cockroach = 2.7, chick = 2.1, mouse = 0.9, housefly = 0.6, pigeon = 0.4, guinea-pig = 0.1.

Clinical Symptoms
Clinically, a bite of Lactrodectus rarely can cause serious health problems. In severe cases, a bite can cause lactrodectism, a disease consisting of raised blood pressure, generalized muscle pain, abdominal cramps, extreme sweating and tachycaia. The average duration of the syndrome in humans is 3 to 6 days. Untreated patients have exhibited clinical signs for a period of 7 days, but weakness and some muscle pain and malaise may persist for weeks. About 75% of the intoxicated patients experience local effects and do not develop systemic envenomation. A pinprick or burning sensation can be felt when bitten by widow spiders. Local pain worsens over time, which may combine with sweating and piloerection. The pain may spread to local lymph nodes or become generalized.

In 25% of the bite incidents, alongside the generic effects, patients experience muscle cramps, spasms and even patchy paralysis. Associated features include nausea, vomiting, headache, fever, malaise, hypertension and tremor.

Treatment
The majority of the patients heal from a bite incident. First aid treatment has not been proven to be useful, but applying pressure and warming the wound may cause symptom worsening.

When leaving patients untreated, symptoms resolve in a couple of hours to days. Respiratory paralysis occurs rarely, and support of the respiratory cardiac tract will be required. When the bite incident becomes too painful, antivenin is administrated., Doctors recommend the use of pain relief medicine before antivenin administration. Such medicines are opioid analgesics, benzodiazepines, calcium gluconate and antispasmodic and relaxant drugs. These medicines are considered because antivenin can induce allergenic reactions. There are cases where systemic latrodectism occur, which can affect the patient for months.

Scientific contribution
αLTX has helped confirm the vesicular transport hypothesis of transmitter release, establish the requirement of Ca2+ for endocytocis, and characterize individual transmitter release sites in the central nervous system. It helped identify two families of important neuronal cell-surface receptors.

The mutant form of αLTX, which is called αLTXN4C and does not form pores, has contributed to research. It helped the approach to deciphering the intracellular signaling transduction mechanism stimulated by αLTX. The mutant toxin can also be used to study the nature and properties of intracellular Ca2+ stores implicated in the toxin receptor transduction pathway and their effect on evoked postsynaptic potentials. The mutant toxin can also be an instrument to elucidate the endogenous functions of αLTX.