Dissertation > Medicine, health > Pharmacy > Pharmacology

To Probe the Interaction Sites between the mSlo1 Pore and the NH2 Terminus of the hβ2 Subunit, and Study on the Mechanism of Desensitization of Pain Receptors TRPV1

Author YaoJing
Tutor DingJiuPing
School Huazhong University of Science and Technology
Course Biophysics
Keywords BK channel TRPV1 channels β2 subunit Point mutations Inactivation Restoration Non-competitive model Capsaicin Adaptive PIP2 Sense organ
Type PhD thesis
Year 2009
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The development of modern life sciences, changing the understanding of the process to life, while the study of ion channels, not only with the signal transmission and transduction closely related, but also related to the molecular mechanisms that induce a variety of genetic and non-genetic diseases. Understanding of ion channels, the fine structure of ion channel gating mechanism and dynamics, to figure out the relationship between the structure and function of ion channels is of great significance. This study consists of two parts: the first part of the large conductance calcium and voltage-activated potassium channel (Maxik channel, also known as BK channel) β2 subunit N-terminal inactivation domain of the α subunit composition of pore sites of action; second part of the article is about the pain-sensitive channel TRPV1 molecular mechanism of accommodation. BK channel exists widely in excitable cells, especially in the nervous system, has important physiological functions of the regulation of intracellular calcium concentration and membrane potential. At present, the voltage-dependent potassium channels (Kv channels) has made great progress. BK channels and Kv channels in the structure and function of both there are some similarities between the various characteristics. As they are combined with β auxiliary subunits, caused by clogging of the hydrophobic amino acid residues of the N-terminal of the β subunit bore N type inactivation. However, the available experimental results show that the bore of the K channel blockers (such as TEA, QX-314, etc.) is able to slow the Kv channel inactivation process, however, can not slow down the inactivation of the BK channel. Why these two are so different? B2 subunit of the N-terminus of the BK channel inactivation domain whether to enter the BK channel α subunit? Both what kind of interaction? Blocker, why not in the pore and inactivation domain in competition binding sites? purpose of this project is one that is to answer and solve these problems that plague us still. We will be the end of the first three hβ2 N amino acids FIW mutation FWI, found that this mutant hβ2 hβ2-FWI BK channel inactivation does not affect channel inactivation recovery curve by a single index into a double exponential. Double exponential using hβ2-FWI recovery characteristics change as the interaction between judgment basis. Upcoming α subunits form channels S6 district hydrophobic amino acids followed by mutation the weak hydrophobic alanine, and then recorded by patch clamp experiments analysis after deactivation channel recovery characteristics change through alanine scanning (Alaninescanning) order to understand the interaction between the two. With the channel pore blockers drugs such as anesthetics QX-314 and TEA further explain the non-competitive mechanism of BK channel inactivation. We got several conclusions: (1) located in the amino acid Ile-323 mSlo1 channel inactivation process inactivation β2 subunit N-terminal domain interaction sites. The two amino acids in the channels of the M314 and V319 are also involved in this process. Subunit of the N-terminal linear hβ2-FWI structural features, we speculate mSlo1 pore hβ2-FWI BK channel inactivation process follows the interaction between :1323-I, V319-W M314-F, which 1323 plays a dominant role. Further clarified inactivation domain of β2 subunit of BK channels indeed entered the bore thereby causing the channel inactivation. (2) channel blockers QX-314 and TEA in the non-specific binding sites in the BK channels. Intracellular blocking agent does not, like in the Kv channel slows channel deactivation process, this is because the the β2 N terminal inactivation domain acting position relatively close to the holes crossing, i.e. the pores within the final one of the hydrophobic amino acids Ile- 323, QX-314 action position is located within the bore, QX-314 does not compete for the same inactivation domain sites of action, which does not affect the β2 caused channel inactivation process. Based on the experimental results, we propose a new non-competitive model, the model can well explain the data, but also to further explain the BK channel structure and gating mechanism provides important experimental and theoretical support. (3) Our results also show that: compared with the wild type mslo1 channel current, mutant 1323A whether single-channel or macroscopic current are significant differences in performance as the wild-type channel does not have outwardly rectifying characteristics. Formal mutant 1323A single-channel single-channel current, very much like the noise current, the same voltage can be recorded simultaneously get several different size means that Ile-323A also cause a change in the conductance of the BK channel. Accordingly, we speculate that the Ile-323 BK channel has a dual role, both the β2 inactivation domain in the pores of the sites of action, while regulation of BK channel gating properties. Also found a very interesting phenomenon, 312 point mutation Leu leucine residues can dramatically change the voltage dependence of the BK channel. Our research group in the subsequent two articles were proved these two presumed: Ile-323 indeed BK channel joint where the 323 amino acid residues, hydrophobic reduced, will make the \channel open when the four subunits of the inconsistencies of interoperability between the change and open probability (Guo et al. Biochem Biophys J.2008 May 1; 94 (9) :3714-25). And Leu312 sites can greatly enhance the voltage sensitivity of the BK channel (Wu et al., In review). Adaptive regulation is a common nature of most of the sensory organs, but pain adaptability of the root causes and the mechanism is still not known. The second part of the article at the receptor level to explore the the the nociceptor TRPV1 channel accommodation of molecular mechanisms. We found that the channel desensitization reaction, only changed the channel agonist sensitivity without affecting the maximum current response, which we proposed in desensitization and gratitude on the basis of pain accommodation concept. The part several main conclusions: (1) Ca 2 via TRPV1 channels open stream cause desensitization reactions, the the desensitization reaction occurs after only changed the channel to capsaicin The sensitivity, the concentration of reduced sensitivity to 14-fold, but this change does not affect the function of the channel. (2) through the joint application of total internal reflection fluorescence microscopy and patch-clamp recording technique, we have confirmed that PIP2 involved in the occurrence of of TRPV1 channels desensitization reaction, the PIP2 decomposition rate and volume changes enough to change the channel of agonist response. With the the drug Rapamycin specifically reduced the PIP2 content within the cells as a research tool, and by measuring the passage of the capsaicin concentration-dependent curve, we further quantify the contribution of PIP2 on desensitization reaction, accounting for about 60%. (3) We also found that the TRPV1 channel agonist sensitivity change depends on the stimulus intensity, stimulus intensity varying degrees of desensitization response. We speculate that there are two possibilities: First, the channel protein and memory function, this possibility is relatively small; TRPV1 channels under different stimulus intensity ion permeability, influx Ca 2 amount and distribution of different characteristics, resulting in a different amount of PIP2 exploded, causing a different degree of desensitization of the reaction.

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