Using Patch Clamp Electrophysiological Methods To Study The Modulation Mechanism Of Peptide Toxins On Potassium Channels

Ion channels are member of membrane protein families, which play important roles in life activity. Mutations of ion channels can lead to ion channelopathy of human, so pharmacological research is very important. Currently, researches of ion channels focus on using X-ray diffraction or solution Nuclear Magnetic Resonance (NMR) spectroscopy to analysis their structures and using patch clamp electrophysiological techniques to study their functions. Animal peptide toxins can interact with ion channels and modulate their open and close states. Therefore, toxins are usually used to study structures and functions of ion channels, and can also be modified to treat diseases. In this thesis, we use protein expression and purification methods to obtain peptide toxins. Then solution NMR and patch clamp electrophysiological experiment are conducted to study the interaction between animal peptide toxins and potassium channels. There are four chapters in this thesis.Chapter 1 introduces the basic knowledge of potassium channels and animal peptide toxins. Potassium channel classification, ion channelopathy, structures of peptide toxins and their interaction with potassium channels are introduced.Chapter 2 briefly describes patch clamp electrophysiological techniques, including patch clamp configuration and derivative technologies, instruments of patch clamp and so on.In chapter 3 we investigate the interaction mechanism between centipede peptide toxin SSD609 and KCNQ1/KCNE1 potassium channel. KCNQ1/KCNE1 channel is closely related to human heart diseases, making its toxins potential drugs. SSD609 from the venom of centipede can act on KCNQ1/KCNE1 potassium channel. Here protein expression and purification methods are used to obtain abundant SSD609. Patch clamp experiments are performed to study its inhibition effect on Iks current. Then solution NMR are conducted to analyze the structure of SSD609. According to the information obtained, we designe the single mutations on KCNE1. Combining the results of patch clamp experiment we conjecture the interaction mechanism between SSD609 and KCNQ1/KCNE1, laying basis for further researches.Chapter 4 introduces the expression and purification of scorpion toxin IbTX and its inhibition effect on BK potassium channel. Heterologous expression of peptide toxin cost less time and money, and the protein can be easily engineered. Fusion tags can facillate the expression of toxins, but the deletion of fusion tags by protease will always introduce extra amino acids to the toxins. Here we use two different proteases to cut off fusion tag from IbTX. Then we test its function by patch clamp experiment and indicate that an extra amino acid will change the function of toxins.