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Functional Impact of Interacting Protein on Kainate Receptors: Necab1 and Neto Proteins

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Functional Impact of Interacting Protein on Kainate Receptors: Necab1 and Neto Proteins Instituto de Neurociencias de Alicante Universidad Miguel Hernandez Thesis manuscript for: PhD in Neuroscience Functional impact of interacting protein on kainate receptors: NeCaB1 and NeTo proteins. Jon Palacios Filardo Supervised by: Prof. Juan Lerma Alicante, 2014 Agradecimientos/Acknowledgments Agradecimientos/Acknowledgments Ahora que me encuentro escribiendo los agradecimientos, me doy cuenta que esta es posiblemente la única sección de la tesis que no será corregida. De manera que los escribiré tal como soy, tal vez un poco caótico. En primer lugar debo agradecer al profesor Juan Lerma, por la oportunidad que me brindó al permitirme realizar la tesis en su laboratorio. Más que un jefe ha sido un mentor en todos estos años, 6 exactamente, en los que a menudo al verme decía: “Jonny cogió su fusil”, y al final me entero que es el título de una película de cine… Pero aparte de un montón de anécdotas graciosas, lo que guardaré en la memoria es la figura de un mentor, que de ciencia todo lo sabía y le encantaba compartirlo. Sin duda uno no puede escribir un libro así (la tesis) sin un montón de gente alrededor que te enseña y ayuda. Como ya he dicho han sido 6 años conviviendo con unos maravillosos compañeros, desde julio de 2008 hasta presumiblemente 31 de junio de 2014. De cada uno de ellos he aprendido mucho; técnicamente toda la electrofisiología se la debo a Ana, con una paciencia infinita o casi infinita. La biología molecular me la enseñó Isa. La proteómica la aprendí del trío Esther-Ricado-Izabella. Joana y Ricardo me solventaron mis primeras dudas en el mundo de los kainatos. Y cada miembro del laboratorio aportó lo suyo. Como primer compañero de fatigas a Jose, luego vinieron Will (que por su culpa me puse gordo, debieron ser las cervezas…), Sergio (al que tuve oportunidad de enseñar parte de mis conocimientos de electrofisiología, pero del cual también he aprendido cosas) y Valeria (la nueva y divertida). También agradezco a Mónica, por su ayuda con las minis, midis, maxis y aportando todo lo que podía. Como he dicho un equipo maravilloso. Lo único que lamento es que con un equipo tan internacional no haya aprendido más idiomas. Pero no todo el tiempo ha sido de tesis, han sido 6 años y las personas que me conocieron cuando llegué a Alicante me comentan que he empezado mi transición de adolescente a hombre. La verdad es que cuando llegué todavía tenía la mentalidad un poco de niño y como se dice, “uno es uno mismo y sus circunstancias”, por ello quería agradecer a todas las personas que hicieron posible todas las circunstancias y evolución que me han llevado hasta aquí. Me llevo muy buenos momentos con muchas personas, y como no quisiera i Agradecimientos/Acknowledgments olvidarme de nadie (porque eso queda fatal), solamente escribiré un, esta frase va por ti, y cada uno ya lo sabe. Mirando al pasado debo agradecer a todos mis educadores, por hacerme como soy, pero en especial a mi familia. A mis padres (aita y ama) por los valores inculcados y a Maialen (hermana), porque aunque siempre nos chinchamos nos queremos mucho. Y ya por fin, gracias mi chica, las palabras más emotivas para ella. Menos mal que las tardes de cortejo dieron sus frutos, porque sino cuando tiempo desaprovechado. Pero lo importante es que lo dieron y espero lo sigan dando. Gracias por aguantarme y comprenderme.Un beso. ii Table of contents Table of contents Agradecimientos/Acknowledgments .................................................................... i Table of contents ............................................................................................... iii Abbreviations and acronyms ............................................................................ vii Work communications ....................................................................................... xi Abstract/Resumen ........................................................................................... xiii I. Introduction ................................................................................................. 1 1. Glutamate receptors ................................................................................. 2 1.1. NMDA receptors .............................................................................. 4 1.2. AMPA receptors ............................................................................... 6 1.3. Kainate receptors ............................................................................. 8 1.3.1. Structure of kainate receptors ........................................................ 9 1.3.2. Pharmacoloy of kainate receptors ............................................... 12 1.3.3. Molecular determinant of kainate receptors cannel gating ......... 14 1.3.4. Allosteric regulation of kainate receptors ..................................... 16 1.3.5. Distribution of kainate receptors .................................................. 18 1.3.6. Interacting proteins with kainate receptors ................................. 19 1.3.6.1. Auxiliary proteins on kainate receptors .............................. 19 1.3.6.2. Transient interacting proteins with kainate receptors ......... 21 1.3.7. Physiological roles for kainate receptors .................................... 26 1.3.7.1. Role of kainate receptors during development ................... 26 1.3.7.2. Role of kainate receptors as mediators of synaptic transmission ........................................................................ 26 1.3.7.3. Role of kainate receptors modulating network excitability ........................................................................... 28 1.3.7.4. Role of kainate receptors modulating synaptic transmission ........................................................................ 28 1.3.8. Kainate receptors facing CNS disorders ..................................... 30 1.3.8.1. Neurological diseases ........................................................ 30 1.3.8.2. Psychiatric diseases .......................................................... 32 iii Table of contents II. Aim ............................................................................................................. 33 III. Material and methods ............................................................................... 37 1. Biological models.................................................................................. 39 2. CDNA constructs .................................................................................. 40 3. Transfections ....................................................................................... 40 4. Electroporations ................................................................................... 41 5. Protein analysis .................................................................................... 42 6. Pull-down experiments ........................................................................ 44 7. Protein immunoprecipitations .............................................................. 45 8. Protein biotinylation experiments ......................................................... 46 9. Immunocytochemistry .......................................................................... 47 10. Bimolecular fluorescence complementation ......................................... 47 11. Real time PCR ..................................................................................... 48 12. Electrophysiological recordings ............................................................ 49 13. Solutions and buffers ........................................................................... 51 IV. Results ................................................................................................. 55 Results I: NeCaB1 and GluK5 containing KARs ...................................... 57 1. Space and time localization of NeCaB1 and GluK5 ................................... 57 2. NeCaB1 interacts with GluK5 containing KARs .......................................... 58 3. Mapping NeCaB1 interaction on GluK5 C-terminal domain .................... 60 4. Ca2+ modulates NeCaB1 and GluK5 interaction ......................................... 62 5. Functional impact of NeCaB1 on GluK1/5 KARs ........................................ 64 6. NeCaB1 increases GluK5 surface expression .............................................. 65 7. Trafficking and affinity modifications by NeCaB1 ........................................ 68 8. NeCaB1 modulates trafficking and affinity of GluK2/5 KARs .................. 71 9. The modulation of GluK2/5 KARs affinity by NeCaB1 is rapid ................ 74 10. Synaptic localization of NeCaB1 ...................................................................... 75 11. Discussion ................................................................................................................ 76 iv Table of contents Results II: Similarities and dissimilarities of KARs tethered to Neto proteins. ........................................................................................................................... 85 1. Neto proteins differentially express during development .......................... 85 2. Neto1 and Neto2 differentially modulate KAR gating ................................ 86 3. Neto proteins increase membrane KARs ...................................................... 88 4. Neto proteins increase affinity of KARs .......................................................... 90 5. Neto proteins alter desensitization recovery rate of KARs ....................... 90 6. Neto proteins
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