Identification of Tentonin 3, a Novel Mechanosensitive Channel and Its Role in Proprioception

학위논문 (박사)-- 서울대학교 융합과학기술대학원 : 바이오제약학과, 2016. 8. 오우택.Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C is activated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The mechanically-activated TTN3 currents were inhibited by knownblockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinatedIntroduction 1 1. Mechanosensation 1 1.1. Somatosensory system 2 1.2. Auditory system 6 1.3. Baroreceptor reflex 8 1.4. Proprioception 9 2. Ion Channels 13 2.1. Overview 13 2.2. Classification of ion channels 14 3. Mechanosensitive ion channels 15 3.1. DEG/ENaC and TRPA1 in Caenorhabditis elegans 18 3.2. MS channels in Drosophila melanogaster 19 3.3. Candidates for MS ion channels in mammal 19 Purpose of This Study 26 Methods 28 1. Molecular cloning of the TTN family 28 2. Cell culture and transfection 30 3. Isolation of DRG neurons 31 4. Channel current recording 32 5. Mechanical stimulation 34 6. Recording of MS afferent activity 34 7. Phylogenetic Analysis 36 8. Antibody production 36 9. Animals and Ttn3 KO mouse 36 10. Immunohistochemistry 37 11. Immunofluorescence 38 12. Western blot 39 13. Inverted screen test 39 14. Beam walking test 40 15. Catwalk Gait analysis 40 16. Statistical analysis 42 Results 44 1. Cloning of Tentonin 3 44 2. TTN3 is required for slowly adapting MA currents in DRG neurons 45 3. Activation by mechanical stimulation. 51 4. Biophysical property 63 5. Putative pore region 75 6. Pharmacology of TTN3 78 7. Expression of TTN3 in DRG neurons 83 8. Expression and functional role of TTN3 in muscle spindles 89 9. Loss of motor coordination in Ttn3 KO mice 94 Discussion 100 References 108 국문 초록 120Docto