Modulation of voltage-gated Na+ and K+ channels by pumiliotoxin 251D: a "joint venture" alkaloid from arthropods and amphibians.

Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa(v)1.2/beta(1), rNa(v)1.4/beta(1), hNa(v)1.5/beta(1) and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK(v)1.1-1.2, hK(v)1.3, hK(v)11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na(+) influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K(+) efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK(v)1.3 was the most sensitive channel, with an IC(50) value 10.8+/-0.5 microM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.

Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, a toxin from the scorpion Odonthobuthus doriae.

Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons. Nav1.7 is found in peripheral sensory and sympathetic neurons and involved in short-term and inflammatory pain. Nav1.8 and Nav1.3 are major players in nociception and neuropathic pain, respectively. In our effort to identify isoform-specific and high-affinity ligands for these channels, we investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion Odonthobuthus doriae. Nav1.3, Nav1.7, and Nav1.8 channels were coexpressed with beta1-subunits in Xenopus laevis oocytes. Na+ currents were recorded with the two-electrode voltage-clamp technique. OD1 modulates Nav1.7 at low nanomolar concentrations: 1) fast inactivation is dramatically impaired, with an EC50 value of 4.5 nM; 2) OD1 substantially increases the peak current at all voltages; and 3) OD1 induces a substantial persistent current. Nav1.8 was not affected by concentrations up to 2 microM, whereas Nav1.3 was sensitive only to concentrations higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of 1127 nM. Finally, the effects of OD1 were compared with a classic alpha-toxin, AahII from Androctonus australis Hector and a classic alpha-like toxin, BmK M1 from Buthus martensii Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to AahII but not to BmK M1, whereas Nav1.8 was affected by neither toxin. In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator of Nav1.7, with a unique selectivity pattern.

Resistance to tyrosine kinase inhibitors: calling on extra forces.

Over the past 5 years, small molecule tyrosine kinase inhibitors have been successfully introduced as new cancer therapeutics. The pioneering work with the ABL inhibitor imatinib (Glivec, Gleevec) was rapidly extended to other types of leukemias as well as solid tumors, which stimulated the development of a variety of new tyrosine kinase inhibitors. Unfortunately, oncogenic tyrosine kinases seem to have little problem to develop resistance to these inhibitors, and there is good evidence that this is not limited to imatinib, but also occurs with other inhibitors, such as FLT3 and EGFR inhibitors. Based on studies with imatinib, mutation and amplification of the target kinase seem to be the most important mechanisms for the development of resistance, but these mechanisms alone cannot explain all cases of resistance. A better understanding of the resistance mechanisms will be required to design improved treatment strategies in the future. In this review, we summarize the current insights in the different mechanisms of resistance to small molecule tyrosine kinase inhibitors, and discuss future improvements that might limit or even overcome resistance.

Molecular basis of the mammalian potency of the scorpion alpha-like toxin, BmK M1.

In-depth structure-function studies of voltage-gated Na+ channels and peptide toxins are continuously increasing our understanding of their interaction. In this study, an effective yeast expression system was used to study the role of 14 N- and C-terminal residues from the alpha-like toxin BmK M1 from the Chinese scorpion Buthus martensii Karsch. With the use of site-directed mutagenesis, all of these residues were individually substituted by one or more amino acids, resulting in a total of 19 mutants. These were then subjected to a bioassay on mice, an elaborate electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5, and para), and a circular dichroism analysis. Our results reveal large mutant-dependent differences that emphasize important and specific roles for the studied residues. By mutating single amino acids, we were able to redirect the alpha-like characteristics of BmK M1 (active on both mammals and insects) to either much higher mammal specificity or, in a few cases, total insect specificity. This study therefore represents a thorough mapping and elucidation of three epitopes that underlie the molecular basis of the mammalian and insecticidal potency of the scorpion alpha-like toxin, BmK M1 on voltage-gated Na+ channels.

Jingzhaotoxin-I, a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation.

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3'- and 5'-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is approximately 30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an alpha-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.

The poison Dart frog's batrachotoxin modulates Nav1.8.

Batrachotoxin is a potent modulator of voltage-gated sodium channels, leading to irreversible depolarisation of nerves and muscles, fibrillation, arrhythmias and eventually cardiac failure. Since its discovery, field researchers also reported numbness after their skin came into contact with this toxin. Intrigued by this phenomenon, we determined the effect of batrachotoxin on the voltage-gated sodium channel Nav1.8, which is considered to be a key player in nociception. As a result, we discovered that batrachotoxin profoundly modulates this channel: the inactivation process is severely altered, the voltage-dependence of activation is shifted towards more hyperpolarised potentials resulting in the opening of Nav1.8 at more negative membrane potentials and the ion selectivity is modified.

Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage.

Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP.

Myasthenic syndrome caused by mutation of the SCN4A sodium channel.

In a myasthenic syndrome associated with fatigable generalized weakness and recurrent attacks of respiratory and bulbar paralysis since birth, nerve stimulation at physiologic rates rapidly decremented the compound muscle action potential. Intercostal muscle studies revealed no abnormality of the resting membrane potential, evoked quantal release, synaptic potentials, acetylcholine receptor channel kinetics, or endplate ultrastructure, but endplate potentials depolarizing the resting potential to -40 mV failed to excite action potentials. Pursuing this clue, we sequenced SCN4A encoding the skeletal muscle sodium channel (Nav1.4) and detected two heteroallelic mutations involving conserved residues not present in 400 normal alleles: S246L in the S4/S5 cytoplasmic linker in domain I, and V1442E in the S3/S4 extracellular linker in domain IV. The genetically engineered V1442E-Na channel expressed in HEK cells shows marked enhancement of fast inactivation close to the resting potential, and enhanced use-dependent inactivation on high-frequency stimulation; S246L is likely a benign polymorphism. The V1442E mutation in SCN4A defines a novel disease mechanism and a novel phenotype with myasthenic features.