Peter N. Strong

Potassium channel toxins.

Many venom toxins interfere with ion channel function. Toxins, as specific, high affinity ligands, have played an important part in purifying and characterizing many ion channel proteins. Our knowledge of potassium ion channel structure is meager because until recently, no specific potassium channel toxins were known, or identified as such. This review summarizes the sudden explosion of research on potassium channel toxins that has occurred in recent years. Toxins are discussed in terms of their structure, physiological and pharmacological properties, and the characterization of toxin binding sites on different subtypes of potassium ion channels.

Identification of two toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channel

Two polypeptide toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium‐activated potassium channels have been identified and partially purified. One toxin, at 50–100 , blocks apamin‐sensitive potassium fluxes in hepatocytes and inhibits [125I]monoiodoapamin binding. The other, more basic, toxin blocks apamin‐insensitive potassium fluxes in erythrocytes at 200 and, to our knowledge, is the first toxin shown to block the erythrocyte calcium‐activated potassium channel with high affinity. The possible co‐identity of this latter toxin with charybdotoxin is discussed.

Antimicrobial peptides from scorpion venoms

Abstract The need for new antimicrobial agents is becoming one of the most urgent requirements in modern medicine. The venoms of many different species are rich sources of biologically active components and various therapeutic agents have been characterized including antimicrobial peptides (AMPs). Due to their potent activity, low resistance rates and unique mode of action, AMPs have recently received much attention. This review focuses on AMPs from the venoms of scorpions and examines all classes of AMPs found to date. It gives details of their biological activities with reference to peptide structure. The review examines the mechanism of action of AMPs and with this information, suggests possible mechanisms of action of less well characterised peptides. Finally, the review examines current and future trends of scorpion AMP research, by discussing recent successes obtained through proteomic and transcriptomic approaches.

Dystrophin and nebulin in the muscular dystrophies

Skeletal muscle from patients with 5 different forms of muscular dystrophy and from 6 fetuses at high risk (95%) for Duchenne muscular dystrophy (DMD) were probed with specific antibodies for the presence of dystrophin and nebulin. Dystrophin was absent in all 5 patients with DMD and 4 of 6 fetuses at high risk for DMD and present in trace amounts in the remaining two. Dystrophin was also undetectable in one borderline DMD/Becker muscular dystrophy (BMD) case and reduced in 2 of 4 cases of BMD. In contrast, dystrophin was present in all 16 biopsies from 4 other types of muscular dystrophy (congenital, limb girdle, Emery-Dreifuss and facioscapulohumeral). Nebulin profiles varied with the type, severity and duration of the dystrophic process. Nebulin was present in 5 of 6 DMD fetal samples but vastly reduced or absent in all samples of clinically manifest DMD.

Characterisation of dystrophin in carriers of Duchenne muscular dystrophy

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in needle biopsy samples taken from the quadriceps muscle of 15 asymptomatic carriers of DMD (13 adults and 2 young girls) and one symptomatic adult carrier. Antibodies to N- and C-terminal regions of dystrophin were used for both Western blot analysis and immunocytochemistry and a monoclonal antibody to beta-spectrin used to assess membrane integrity. All asymptomatic adult carriers showed some abnormality in dystrophin immunostaining but very few negative fibres were present. A clear mosaic of dystrophin positive and negative fibres was seen only in the adult symptomatic carrier and the two young girls. On a Western blot, all carriers studied had dystrophin of normal molecular weight, but most had reduced abundance. In adult carriers, the amount of dystrophin relative to normal controls varied, but it was unrelated to age, serum creatine kinase (CK) levels or to the degree of pathology. Carriers with normal CK showed abnormalities in dystrophin expression. The dystrophin immunoblotting profile of the 2 young girls was very similar to that of their mothers, but the mosaic pattern of immunostaining was not apparent in the older carriers. In conclusion, dystrophin immunostaining and Western blot analysis of biopsy samples from asymptomatic carriers is often abnormal and they may be useful additional aids for establishing carrier status, particularly in younger girls.

Interactions between dendrotoxin, a blocker of voltage-dependent potassium channels, and charybdotoxin, a blocker of calcium-activated potassium channels, at binding sites on neuronal membranes

Dendrotoxin I (DpI) from black mamba venom (Dendroaspis polylepis) has high affinity binding sites on rat brain synaptic membranes. Native DpI displaced [125I]-DpI binding with a Ki of 1 x 10(-10) M, and over 90% of specific binding was displaceable. Charybdotoxin isolated from the Israeli scorpion venom (Leiurus quinquestriatus hebraeus), also displaced [125I]-DpI binding, with a Ki of approximately 3 x 10(-9) M, although the displacement curve was shallower than with native DpI. Both toxins are thought to be high affinity blockers of specific K+ currents. Charybdotoxin selectively blocks some types of Ca2+-activated K+ channels, whereas dendrotoxins only block certain voltage-dependent K+ channels. The interaction between the two types of toxin at the DpI binding site is unexpected and may suggest the presence of related binding sites on different K+ channel proteins.

Effects of potassium channel toxins from Leiurus quinquestriatus hebraeus venom on responses to cromakalim in rabbit blood vessels

1 The effects of fractionated Leiurus quinquestriatus hebraeus venom on cromakalim‐induced 86Rb+ efflux in rabbit aortic smooth muscle were examined. 2 Crude venom (0.1–30 μg ml−1) produced a concentration‐dependent decrease of 1 μm cromakalim‐induced 86Rb+ response. The maximum blocking activity attainable was approximately 60%. 3 Fractionation of crude venom by gel permeation chromatography and subsequent chromatography on a cation ion‐exchange column, produced two fractions (X and XI), active in the 86Rb+ blocking assay. 4 Fraction XII contained charybdotoxin (∼85% pure). After a final high performance liquid chromatography (h.p.l.c.) purification step, the purified toxin failed to inhibit the cromakalim‐stimulated 86Rb+ efflux although it was a potent inhibitor of A23187‐induced K+ flux in human erythrocytes and the large conductance calcium‐activated potassium channel in rabbit portal vein smooth muscle. 5 Subsequent purification of fraction X by h.p.l.c. yielded a minor peak which contained 86Rb+ blocking activity. This subfraction was also capable of inhibiting apamin‐sensitive, angiotensin II‐stimulated K+ flux in guinea‐pig hepatocytes. 6 It is concluded that the potassium channel opened by cromakalim in rabbit aortic smooth muscle is not blocked by charybdotoxin but by another distinct toxin in the venom of Leiurus quinquestriatus hebraeus.

High affinity binding of [125I]monoiodoapamin to isolated guinea‐pig hepatocytes

The bee venom neurotoxin apamin has been labelled with 125I and its binding to isolated guinea‐pig hepatocytes measured under physiological conditions. A single saturable component of [125I]monoiodoapamin binding with a K d of 350 pM and B max of 0.99 fmol/mg dry wt was identified. Native apamin displaced labelled apamin with a K d of 376 pM which is broadly in keeping with the concentrations found to inhibit K loss from guinea‐pig hepatocytes. These observations, together with the binding found in other tissues, suggest that specific binding of labelled apamin is particularly associated with apamin‐sensitive, Ca‐activated K‐channels.

A convenient bioassay for detecting nanomolar concentrations of tetrodotoxin

We describe a convenient as well as highly sensitive bioassay procedure for detecting nanomolar concentrations of tetrodotoxin. The method utilizes desheathed frog (Rana pipiens) sciatic nerve mounted in a modified sucrose gap apparatus and depends on a measure of the reduction of the compound action potential height when the nerve bundle is exposed to the test solution (ca. 1 ml). The concentration of TTX needed to effect a 50 per cent reduction in height of the compound action potential was found to be 8 nM. We have shown that the Oregon newt Taricha granulosa contains on the order of 1 mg of TTX per g of newt.

Equine muscular dystrophy with myotonia.

OBJECTIVES To describe a case of equine muscular dystrophy with myotonia. METHODS A 5-year-old horse presented with hypertrophy and delayed relaxation of the muscles of the hindlimbs from age 2 months. Testicular atrophy developed from 2 years of age. Action and percussion myotonia was associated with weakness in these muscles, and EMG showed diffuse myotonic discharges and myopathic features. Biopsy of the gluteal muscle showed adipose and connective tissue infiltration, marked variation in muscle fibre size, and moth-eaten, ring and whorled fibres. RESULTS Injection of apamin, a peptide blocker of calcium-activated potassium channels, which inhibits myotonia in human myotonic dystrophy, was ineffective in blocking myotonic discharges. Discharges promptly abated with 2% lidocaine injection. CONCLUSIONS Myotonia in this horse is associated with dystrophic changes similar to human myotonic dystrophy, though there are some pharmacological differences.