K+ currents in isolated vestibular afferent calyx terminals.

Vestibular hair cells transduce mechanical displacements of their hair bundles into an electrical receptor potential which modulates transmitter release and subsequent action potential firing in afferent neurons. To probe ionic mechanisms underlying sensory coding in vestibular calyces, we used the whole-cell patch-clamp technique to record action potentials and K(+) currents from afferent calyx terminals isolated from the semicircular canals of Mongolian gerbils. Calyx terminals showed minimal current at the mean zero-current potential (-60 mV), but two types of outward K(+) currents were identified at potentials above -50 mV. The first current was a rapidly activating and inactivating K(+) current that was blocked by 4-aminopyridine (4-AP, 2.5 mM) and BDS-I (up to 250 nM). The time constant for activation of this current decreased with membrane depolarization to a minimum value of approximately 1 ms. The 4-AP-sensitive current showed steady-state inactivation with a half-inactivation of approximately -70 mV. A second, more slowly activating current (activation time constant was 8.5 +/- 0.7 ms at -8 mV) was sensitive to TEA (30 mM). The TEA-sensitive current also showed steady-state inactivation with a half-inactivation of -95.4 +/- 1.4 mV, following 500-ms duration conditioning pulses. A combination of 4-AP and TEA blocked approximately 90% of the total outward current. In current clamp, single Na(+)-dependent action potentials were evoked following hyperpolarization to potentials more negative than the resting potential. 4-AP application increased action potential width, whereas TEA both increased the width and greatly reduced repolarization of the action potential.

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail.

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus.

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.

Purification and characterization of a short insect toxin from the venom of the scorpion Buthus tamulus.

A short chain peptide has been isolated from the venom of a red scorpion of Indian origin, Buthus tamulus. This peptide was purified using ion exchange and reverse phase chromatography and was characterized by molecular weight determination and amino acid sequence. The primary structure analysis shows that BtITx3 is a short peptide of 35 amino acid residues having a molecular weight of 3796 Da. The toxin shows toxicity towards the Lepidopteran species of insect Helicoverpa armigera causing flaccid paralysis and even death within 24 h. It shows more than 50% homology with the short insectotoxins having four disulfide bridges, which suggests that the toxin belongs to the class of short chain toxins blocking the chloride ion channels. This sequence homology study has also helped to bring out the structure-function relationship between the various short toxins. Homology modeling done by using template structure of a known toxin indicated that this toxin consists of a similar alpha/beta scaffold, as present in other scorpion toxins.