Study on the Connexion 32 and its role in the release of ATP.

• Autores: Mª Eugenia Grandes Vilaclara
• Directores de la Tesis: Joan M. Blasi Cabús (dir. tes.), Carles Solsona Sancho (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2008
• Idioma: inglés
• Tribunal Calificador de la Tesis: Francisco Ciruela Alférez (presid.), Artur Llobet Berenguer (secret.), Eduard Serra Arenas (voc.), Luc Leybaert (voc.), Victor Volpini Bertrán (voc.)
• Materias:
  • Ciencias de la vida
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • The aim of this 4 year long work was to study the Connexin32 (Cx32), a protein that forms hemichannels anchored in the plasma membrane. Two hemichannels of adjacent cells form a gap junction. Mutations in Cx32 have been associated to the X-linked form of Charcot-Marie-Tooth disease, a neurodegenerative illness affecting the peripheral nervous system. Cx32 is expressed in Schwann cells, in the paranodal zones. Using Xenopus laevis oocytes to express Cx32 hemichannels, we have monitored simultaneously the release of ATP and the ionic currents. ATP in the medium was detected with luciferin-luciferase reaction and the ionic currents were recorded under two electrode voltage clamp. Depolarization of oocytes expressing Cx32, from -40mV to +80mV induced an outward current and at the end of the pulse a transitory peak of ATP release. Using Cx32 transfected HeLa cells, we captured the luminescence due to ATP release, when cells were under a hypotonic solution containing luciferin-luciferase mixture. Isotonic solution was 280 mOsm and hypotonic solution was 150 mOsm. We also studied the release of ATP in Schwann cell cultures and again the hypotonic conditions induced a rapid increase of extracellular ATP. Finally, when we repeated the voltage clamp experiments expressing Cx32 and also syntaxin 1A, we saw a partial inhibition that of the currents and the release of ATP, which didn't happen when we repeated the hypotonic shock with HeLa cells expressing Cx32 and syntaxin. In order to keep on working in the ATP release through Cx32 hemichannels and its relation with CMT disease five different mutation already described in CMTX patients were generated in the lab, to study the ATP release through Cx32 mutated hemichannels in TEVC experiments with oocytes and transfecting mammal cells. This last part is not finished but it is conyinued by a predoctoral student of Dr. Solsona.