Optogenetics uses light-regulated ion channels and pumps to modulate the activity of neurons with high temporal precision. To date, inhibition of neuronal firing is obtained by means of hyperpolarizing pumps, and/or chloride channels. However, a light-gated potassium (K+) channel would represent a more physiological tool because of the universal role of K+ in repolarizing membrane potential in animal cells. We recently engineered a synthetic light-gated channel by fusing the LOV (light oxygen voltage) domain of the plant blue-light receptor AsPhototropin1 to the viral K+ channel Kcv-PBCV1. The resulting channel BLINK1 (Blue Light Induced K+ channel) is reversibly activated by blue light and shows K+ selectivity and high single channel conductance, when expressed in HEK 293T cells. However, BLINK1 low expression rate at the plasma membrane (PM) (less than 10% in HEK 293T cells) prevented so far its application in optogenetics. The goal of my PhD thesis has been to improve BLINK1 expression in HEK293T cells and to test it in two model systems, zebrafish and mouse. To this end, I have applied molecular and cellular approaches for modifying the trafficking and the overall folding and stability of the channel. Both the LOV domain and the pore domain have been modified by either the addition of PM anchoring motifs and structural elements (LOV domain) or the addition of trafficking motifs (Kcv). The outcome of this study, BLINK2, was obtained by adding a 14-3-3 signal peptide to the C terminus of BLINK1. It showed a moderate increase in PM expression (26% vs 10%) compared to BLINK1, but responded to light with a delay of about 90 s. We further improved BLINK2 by introducing a point mutation (Q513D) in the LOV domain that reduced the delay to about 30 s. When tested in mouse, BLINK2 Q513D efficiently inhibited spontaneous firing in brain slices. BLINK2 Q513D is currently undergoing a test in vivo in a mouse behavioral experiment.
|Titolo:||IMPROVING TRAFFICKING AND KINETICS OF A SYNTHETIC LIGHT-GATED POTASSIUM CHANNEL|
|Data di pubblicazione:||26-giu-2017|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||IMPROVING TRAFFICKING AND KINETICS OF A SYNTHETIC LIGHT-GATED POTASSIUM CHANNEL / L.s. Alberio ; scientific tutor: A. Moroni. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2017 Jun 26. ((29. ciclo, Anno Accademico 2016.|
|Digital Object Identifier (DOI):||10.13130/alberio-laura-silvia_phd2017-06-26|
|Appare nelle tipologie:||Tesi di dottorato|