Study Of Functional Regulation Of Kir Channels

Inwardly rectifying potassium (Kir) channels comprise a super family composed of seven subfamilies (Kir 1-7) containing about 20 members in mammals. Kir channels are expressed in many tissues. The Kir currents show the inward rectification property that means potassium ion can flow into the cell more easily than flow out. Kir channels serve important roles in cellular physiology such as cell excitability and K+ homeostasis. Besides these common functional properties, each subfamily of Kir has its own characteristic. Kir1.x is involved in transepithelial membrane transport of H+ and K+, particularly in the kidney. Kir1.x is exquisitely sensitive to changes in intracellular pH in the physiological range. It is closed down by acidification, while alkalinization results in channel opening. Kir2.x is predominantly expressed in heart, skeletal muscle and nervous system. Kir2.x plays a role in controlling the excitability of heart and brain. Kir3.x is expressed in cardiac myocytes and central neurons. Kir3.x channels are G-protein-activated and mediate the effects of certain G-protein-coupled receptors on electrical activity in cardiac, neuronal and neurosecretory cells. The ATP-sensitive K+ channels(KATP), composed of Kir6.x and SUR subunits, are regulated by cytosolic nucleotides. They are important in the regulation of insulin secretion and the protective response to cardiac and cerebral ischaemia. The physiological roles of Kir4.x, Kir5.x and Kir7.x channels have not been fully elucidated. A common feature of Kir channels that has emerged recently is that they all require the membrane phospholipid, PIP2 (phosphatidylinositol 4,5-bis-phosphate) to maintain their activity. Furthermore, the activity of Kir channels can be regulated by many other factors, while each Kir channel is regulated by these factors diversely. We expressed Kir channels and Kir modulation proteins in Xenopus oocytes or COS cells to study the Kir regulation by different modulator.The expression of Kir channels in Xenopus oocyte and the properties of Kir channelsAim: To express Kir channel in Xenopus oocytes and record the currents of Kir channels through two-electrodes voltage clamp technique.Methods: ①Transcription of all Kir channel in vitro: All cDNA constructs were subcloned into the pGEMHE plasmid vector and the sequence of all constructs were confirmed by sequencing. All cRNAs were transcribed using RibomaxTM Large Scale RNA Production Systems-SP6 and T7 Kit after linearizing the DNA constructs with appropriate restriction endonuclease. ②Preparation of oocytes: Xenopus oocytes were surgically extracted, dissociated, defolliculated by collagenase (2mg/ml) for 1.5-2 hours, then washed with ND96 solution. ③ Microinjection of oocytes: Each oocyte were injected with 50nl of water containing the desired cRNA. cRNA of the various Kir channels and their mutants and of receptors was injected in the range of 0.5-10 ng/ oocyte depending on the functional expression level of the given construct.④ Current recording: Whole cell currents were recorded under two-microelectrodes voltage clamp using Gene clamp 500B amplifier.Results: ①Analysis of linearized plasmid DNA by agarose electrophoresis: A proper restriction enzyme was chosen to cut the circular plasmid from the downstream of the insert cDNA. The circular plasmid DNA showed more than one bands in the electrophoresis, while the linearized DNA showed only one band. ② The analysis of in vitro transcribed cRNA: RNA has secondary structure and gave a smeared band on the gel. We used RNA sample buffer containing deionized formamide and formaldehyde to denature RNA, in order to get a clear RNA band on the eletrophoresis. ③ Electrophysiological experiment: Kir channel currents can be recorded using two-electrodes voltage clamp method at 2~3 days after microinjection of oocytes. The voltage was changed from –120mV or –100mV to +80mV gradually. The current-voltage relationship (I-V) curve of Kir channel current showed significant inward rectifier property (the potassium ion...