The Role Of Hyperpolarization Activated Cyclic Nucleotide Gated Cation Channel In The Control Of The Excitability Of RIN-14B Cells

Enterochromaffin cells are diffusely distributed within the gut epithelium and more than 90%5-HT within the body is synthesized and released by EC cells.The5-HT receptor family consists of 7 subtype receptors,named 5-HT_1-7.Most of them is G-protein-coupled receptor but only 5-HT₃ is Ion channel receptor.5-HT released by EC cells can play a major role in the regulation of gastrointestinal function and other organ systems by paracrine,autocrine and blood circulation.Hyperpolarization-activated cyclic nucleotide-gated cation channel plays a major role in spontaneous depolarization process of autorhythmic cell which can control heart pacing.HCN channel family consists of 4 cation channels,named HCN1-HCN4.Functional HCN channels are heterotrimeric or homotrimeric channels and differ in channel sensitivity and kinetics to cAMP.In cells expressing HCN channels,membrane hyperpolarization to about-60mV can induce an inward current（Ih）,which make the membrane depolarization and then increase the cell’s excitability.Our group found that EC cells high express HCN2 channels and in the isolated mouse small intestine,adding exogenous ZD 7288（HCN blocker）caused an decrease in 5-HT outflow.On the other hand,in the isolated mouse small intestine,adding exogenous cAMP or cholera toxin（increase endogenous cAMP）can both increase in 5-HT outflow.These suggest that HCN2 channel drive 5-HT release.So the aim of current study is to research the role of HCN channel in control of the excitability of EC cells.We use EC cell’s model RIN-14B cell at the same time we research it to provide more information for those who research EC cells.1.The electrophysiological characteristics of RIN-14B cellRIN-14B cells,a rat pancreatic islet cell line,which are considered to be a model for EC cells can secrete 5-HT.Little is known about RIN-14B cells,so to make sure the electrophysiological characteristics and the function of HCN channels of RIN-14B cells we used current clamp to record the membrane potential.we found a inward current stimulation can evoke action potential which suggest that RIN-14B cells are excitable cells.TTX 1μM,0.1μM both abolish action potential but 200μM and 5mM CdCl2 have no effect.So the action potential is mediated by Na channels.2.Functional HCN channel is expressed in RIN-14B cellFirst we use voltage-clamp recording in RIN-14B cells.We recorded an voltage-dependent current which would be inhibited by ZD 7288（HCN blocker）under-60mV to-120mV voltage clamp.Then we found a typical“sag”membrane potential which would also be inhibited by ZD 7288 under-140pA current-clamp recording.These results suggest that RIN-14B Cells express functional HCN channels.Ivabradine is used to cure heart failure by blocking HCN channel of cardiac autorhythmic cells in clinic.We found that 10μM,30μM,100μM Ivabradine inhibit the“sag”membrane potential in concentration-dependent of RIN-14B cells under-140pA,600ms hyperpolarization current.3.HCN channel control the excitability of RIN-14B cellswe recorded that 30μM ZD 7288 and 30μM Ivabradine both can make the membrane potential of RIN-14B cell hyperpolarization in resting state.This indicated that HCN channel induces an inward current which make the membrane depolarization in cell resting state.Then,we found a rebound depolarization or a rebound depolarization spike which is a response to the offset from hyperpolarization after a-140pA outward current stimulation.HCN channel blocker not only increased the latency of RD and decreased the amplitude of RD but also inhibited the firing of RD spike.The results suggest that HCN channel increase the cell excitability and then increase RD evoking.4.In inflammatory state,the excitability of RIN-14B cell and functional HCN channels expression increase.We then attempted to reveal the possible role of HCN channels in inflammatory state.After TNF-α500pg/ml treatment,the action potential firing rate and probability significant increase under depolarization stimulate.The“sag”membrane potential was more obvious in inflammatory under-140pA current stimulation and Ivabradine would also inhibit the sag.In summary,this study investigated the possible role of HCN channels in regulation of excitability of RIN-14B（EC）cells in normal and inflammatory state at the same time we provide method and information for those who want to use RIN-14B cells.Our results showed that functional HCN channels are expressed in RIN-14B cells and induce an inward current（Ih）,which tend to increase the cell’s excitability.In inflammatory state,functional HCN channels expression and the excitability of RIN-14B cell increase.These support that HCN2 channel drive 5-HT release which would provide a new target for disease treatment.