High resolution tagging for ion channels in neural membrane

The major aim of this project is to develop novel chemical tagging methods for quantitative electron microscopy which will enable us to count absolute number and subunit composition of ion channels on neuronal cells in the brain. In the last decade, green fluorescent protein and its derivatives opened up new fields in visualization of biomolecules at the light microscopic level. However, there are few such tags for electron microscopy (EM) and none of them has one-to-one resolution to visualize all single molecules in situ. To understand how neuronal cells receive chemical and electrical inputs and integrate them to process information in the brain, precise localization and quantification of numbers of various ion channels as well as subunit composition of individual channels on neuronal plasma membrane are essential. In this project, we will combine freeze-fracture replica with 1-2 nm metal particle labeling methods in Shigemoto lab and reactive tag systems in Ojida lab to develop new EM tag methods for counting absolute numbers of ion channel subunits. New synthetic chemical probes that bind to specific tag sequences will be applied to various subunits of ion channels such as voltage dependent potassium and calcium channels expressed in the cerebral cortex by in utero electroporation and knock-in technology in mouse. The chemical probes will be visualized with 1-2 nm ultrasmall gold and palladium particles at the EM level as well as fluorescent dyes at the LM level to identify distribution and number of different metal particles in distinct membrane compartments of pyramidal cells. These ultrasmall gold and palladium particles can be visualized by 200kV scanning transmission electron microscope and differentiated by highly sensitive energy-dispersive X-ray spectrometry (EDS). The particle labeling will be calibrated with electrophysiological measurements of ion channel numbers to prove one-to-one detection sensitivity of the method. We will apply this method to count the numbers of Kv and Cav channels and subunit compositions at the single channel levels in dendritic and axonal plasma membranes. The results obtained will be fundamental information for the analysis of integration of chemical and electrical signals in neurons and its simulation for understanding neuronal computation in the brain. The newly developed EM tag methods are also expected to be very useful for wider biological applications for visualization and quantification of other membrane proteins.

The major aim of this project is to develop novel chemical tagging methods for quantitative electron microscopy, which will enable us to count absolute number of ion channels and receptors on neuronal cells in the brain to obtain fundamental information for understanding neuronal computation in the brain. In the last decade, green fluorescent protein and its derivatives opened up new fields in visualization of biomolecules at the light microscopic level. However, there are few such tags for electron microscopy (EM) and none of them has one-to-one resolution to visualize all single molecules in situ. We combined freeze-fracture replica labeling methods developed in Shigemoto lab and reactive tag systems developed in Ojida lab to establish a new EM tag method. New synthetic chemical probes that bind to a specific tag sequence were first applied to a potassium channel and bradykinin receptor expressed in HEK cells. The chemical probes were visualized at the EM level with 1-2 nm ultrasmall gold and silver intensification. The results showed a high sensitivity of the method compared with conventional immunolableing for green fluorescent protein, and will be applied for neuronal membranes, and also expected to be very useful for wider biological applications for visualization and quantification of other membrane protein complexes.