"voltage-gated calcium channels", "calcium-activated ion channels", "Clustering", "Super resolution microscopy"
The neuronal cells generate the action potentials (APs) over a far wider range of frequencies, amplitude, and duration. Also, neurons can respond to small changes in input currents with significant changes in firing frequency. This diversity of neuronal APs can be attributable to the specified sets of ion channels in the neuronal cells. For example, voltage-gated calcium channels (CaV channels) regulate calcium influx responding to the transmembrane voltage changes. The changes in intracellular calcium concentration control the various cellular proteins and physiological processes. A calcium-activated K+ channel, BK channel, is one of such cases to play an essential role in feedback control of calcium influx and cell excitability. A recent study revealed that CaV1.3 and BK channels are proximally clustered and functionally coupled in the cultured neurons by using super-resolution imaging and electrophysiological recording techniques. To investigate the CaVs and calcium-activated ion channels’ clustering patterns at the super-resolution level, we firstly screened antibodies specific to the different channels. We have examined the expression patterns of the various combination of the voltage-gated calcium channels (CaV1.3, CaV2.1, CaV2.2, CaV2.3, CaV3.2, and CaV3.3) and calcium-activated ion channels (Bestrophin-1, Ano-1, BK, and SK channels) in the hippocampal neurons. For this screening purpose, expression patterns were examined under the confocal microscope and structured illumination microscopy (SIM) imaging. From the confocal and SIM images, we found that some ion channel combinations can be assigned as “co-localization” and others as “non-colocalization”. SIM image was obtained quantitative results through the Nearest-neighbor distance method. The combination of channels could be screened by the image distribution pattern and distance analysis method. The channel combination will be approached in an electrophysiological method to confirm proximal clustering.
Table Of Contents
Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 5 2.1 Cell culture and cDNA Transfections 5 2.2 Primary neuron culture 5 2.3 Immunofluorescent Staining 5 2.4 Acquisition of confocal and SIM imaging 7 2.5 Nearest-neighbor distance analysis of N-SIM images 7 2.6 Plasmids and Antibodies 7 2.7 Ion channel patch-clamp recordings 8
Ⅲ. RESULTS 12 3.1 Antibody screening for detecting ion channels 12 3.2 Ion channel distribution patterns monitored under confocal microscope 16 3.3 Distribution patterns of CaV and BKCa channels expression under SIM 20 3.4 Distribution patterns of CaV and SKCa channels expression under SIM 25 3.5 Electrophysiological recording of CaV and BKCa channels 33 Ⅳ. DISCUSSION 39 Ⅴ. APPENDIX 42 Ⅵ. REFERENCES 44 Ⅶ. Abstract in Korea (요약문) 47