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Effect of IVM on rP2x4 Channel Deactivation

Author: YuQiaQia
Tutor: LiZhiYuan
School: University of Science and Technology of China
Course: Biochemistry and Molecular Biology
Keywords: ATP P2X4Rs IVM ATP binding ectodomain deactivation timeτoff patch-clamp techniques
CLC: Q25
Type: Master's thesis
Year: 2011
Downloads: 6
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Abstract

P2X receptors are membrane non-selective cation channels, which are gated by extracellular ATP (adenosine triphosphate). Because P2X receptors are widely distributed in both excitable and non-excitable cells of vertebrates, P2X receptors mediate many physiology processes including inflammation, neurotransmitter releasing and neuropathic pain.IVM (Ivermectin), a kind of large macrocyclic lactone, can specifically augment the current amplitude and significantly prolong the deactivation time (τoff) of P2X4Rs (P2X4 receptors) rather than other types of P2XRs.It was reported by Khakh and his colleagues that high concentration of IVM can not only increase the size of the currents of P2X4Rs, but also prolong the deactivation time of P2X4Rs after ATP removal, on the contrary low concentration of IVM failed to influence the channel deactivation. Previous research by Silberberg indicated that IVM binding to the receptor can be affected allosterically; binding site of IVM to the receptors is a hydrophobic crevice which was formed between the phospholipid and TMs of one subunit; the sites of ATP and IVM binding to the receptor were not overlapped. Based on their works, we proposed the hypothesis as following: 1, each P2X4 receptor may combine with one IVM molecule in the presence of low concentration of IVM while two or three IVM molecules during applying high concentration of IVM, which would have the effect of stabilizing the ATP binding to P2X4 receptors; 2, high concentration of IVM plays the role of stabilizing the ATP binding to receptors indirectly, part ectodomain near TMs is responsible for this effect; 3, phenylalanines possessing bulky side chains in that region may be the residues involved in the effect of high concentration of IVM.In this study, we aimed to identify the potential phenylalanine residues which located in ectodomain nearby the TMs, that contributing to the IVM effect on channel deactivation; HEK293T was used as the model cells to express the ratP2X4Rs protein. We used the reverse-PCR technique to introduce alanine point mutation into ratP2X4 WT channel and recorded the electrophsiological data with whole cell recording model of patch-clamp techniques. F200, F319 and F330 were selected as the sites of alanine mutagenesis.Our results indicated that all mutants formed functional ion channels. Compared with WT, F319A mutant’sτoff was nearly unchanged and F200A had a reducedτoff while F330A had an increasedτoff in the presence of high concentration of IVM. In addition, F200A and F330A mutants didn’t change the ATP potency and F330A mutant stabilized the IVM binding to ratP2X4Rs. These results suggested that F200, which is a residue of ectodomain nearby TMs, was responsible for the effects of high concentration of IVM on ratP2X4Rs channel deactivation. F330 contributed to IVM binding to channel, and it needs to be verified whether F330 is a potential site for the effect of high concentration of IVM on channel deactivation by measuring the EC50 of ATP on F330A in the presence of high concentration of IVM.

Effect of IVM on rP2x4 Channel Deactivation

Abstract

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