Functional Analysis Of Membrane Proteins Based On Patch Clamp Technique And Fluorescence Lifetime Imaging Microscope

Membrane proteins are widely distributed in various types of cell membranes and participate in a variety of life activities,so they are one of most important research objects.The function of membrane proteins depends on the structure,and the structure regulates the function.Meanwhile,conformational changes will occur during the function of membrane proteins.Therefore,we are very interested in the structure,function and dynamic changes of membrane proteins.For structural analysis,Crystallography and Cryoelectron Microscopy(CryoEM)are effective methods to gather high-resolution information,while the patch clamp technique and fluorescence lifetime imaging microscopy(FLIM)are effective means to explore the function.We often combine patch clamp or FLIM with the analysis of structure to explore the function and dynamics of membrane proteins.Patch clamp technique is a method to analyze the function of ion channels by recording the electrical signals of ion channels on the membrane.In this paper,I compared the opening and closing time and current of human acid-sensing ion channel hASICla by patch clamp technique,and analyzed the ion selectivity and gating mechanism.Explore the binding site and mechanism of Mambalgin-1 and provide a screening way for new drug research and development.To compare the differences between hASICla and cASICl,explore the regulation mechanism of Mambalgin-1,and analyze the structure and function of hASICla truncations.By mutating the possible binding sites of hASICla and Mambalgin-1,they were found that the binding sites of hASICla and Mambalgin-1 were mainly located in the thumb domain of hASICla and Finger I and Finger II of Mambalgin-1.When hASICla was in closed state,Mambalgin-1 could be bound to reduce proton sensitivity,so as to achieve the inhibitory effect.At the same time,the difference of activation mechanism between hASICla and cASICl lead to the difference of Mambalgin-1 inhibition.To explore the function of the N/C-terminal truncation of hASICla,we found the importance of the N/C-terminal of hASICla,which involved the conformational changes from desensitization to close state as well as the expression.Fluorescence lifetime imaging Microscopy(FLIM)has the ability of high resolution and high sensitivity to detect protein and its microenvironment.In this study,we fused EGFP into the C-terminal of ferroportin(FPN)and regulated its transport by manganese ion.Compared with the fluorescence intensity imaging which is greatly affected by the concentration of the fluorophore,we found that the fluorescence lifetime imaging technology detected the characteristics of the fluorophore itself through the time domain or frequency domain methods,and was not affected by the concentration,light path scattering and bleaching quenching,so it has excellent spatial resolution and measurement accuracy.The two peaks of fluorescence lifetime before and after Mn2+treatment were consistent with the two conformational states reported by previous crystallographic analysis.Therefore,FLIM can distinguish the conformational subpopulations of membrane proteins reliably and effectively.Indeed,we are expected to combine patch clamp technique with FLIM.Patch clamp technique can obtain the open,desensitized and closed states of ion channels.If we can analyze the fluorescence lifetime at the same time to detect the local conformational changes of proteins in the fluorescent site,it would be better for structure,function and dynamics research of ion channel.