Prokaryotic Expression And Characterization Of A Novel Potassium Ion Channel Inhibitor Lam-Z1in The Salivary Gland Of Firefly

The firefly is the luminous insects belonging to Insects Coleopteran Lampyride which widely distributed in tropical and substropical and temperate region. It is a completely abnormal development beetle, that experiences four periods of egg, larva, pupa and adult. The larva will inject venom into snail using the palate. After the snai completely palsied, the firefly will release digestive juices to dissolve the snai and then inhale. Therefore, through the way of life we deduce that there will be some neurotoxins in the salivary gland of firefly.The ion channel is a special transmembrane protein. In the centre of the channel there is a pore which allows ion to pass through the membrane by means of switch. It plays an important role in the signal transmission between intracell and extracell. The ion channel is selective and controllable. According to the ion channel mechanisms, the ion channel can be divided into voltage-gated ion channel, ligand-gated ion channel and mechanically-gated ion channel. The voltage-gated ion channels consist of sodium ion channel, potassium ion channel, calcium ion channel and chloride ion channel. The ion channels studied in this paper belong to the voltage-gated ion channel.Lam-Z1is a polypeptide cloned from the cDNA library of the firefly’s salivary glands. We found that the polypeptide has22%～34.2%homology with the potassium ion channel inhibitor by amino acid sequence comparison.In order to further study the structure and function of peptide, we used the PET expression system of E.coli to express Lam-Z1successfully. Through the optimizing we found the best induction condition that the expression efficiency was improved significantly. Moreover, the expression quantity of the recombinant protein was32.2%. we got purified peptide by Sephadex G-50and RP-HPLC etc. Using whole cell patch clamp to detect the function of Lam-Zl to ion channel, we found that Lam-Zl has no apparent effect on sodium ion channel and calcium ion channel, while it can inhibit potassium ion channel, and the IC50is1.34μM±0.2μM. The inhibition don’t rely on the open state of channel. Lam-Z1combines the voltage sensor, decreases the voltage sensitivity and increases the activation voltage of the channel, which leading to the Ⅰ-Ⅴ curver migrate20mV±2mV to the right. In addition, Lam-Zl changes the related features of dynamics, resulting in the steady-state inactivation curve in a depolarization direction, slowing done the inactivation schedule.There are few reports about the salivary gland of firefly, we found a kind of potassium ion channel inhibitor from the salivary gland of firefly for the first time.