BLINK1 is a synthetic light-gated potassium (K+) channel reversibly activated by blue light, encoded by a single gene, whose activity does not need the addition of cofactors. Transient ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed two-days-old Zebrafish larvae, confirming in vivo applicability of BLINK1 as a single-component optogenetic tool that can establish sustained, physiological hyperpolarization of cells at K+ electrochemical potential (EK). In HEK293 cells, we record a measurable BLINK1 current in less than 10% of the transfected cells. Immunolocalization experiments confirmed a very low frequency of BLINK1-specific signal on the plasma membrane (PM) of transfected cells and retention of the protein in inner cellular compartments. To fix this problem, we have added to the channel C-terminal region diacidic ER export signals from other K+ channels (mKir2.1, KAT1), plasma membrane trafficking sequences (YXXΦ motifs) and mode III 14-3-3 binding sites found in other channels and pumps (TASK channels, MHA2 H+-ATP-ase, KAT1). In most cases, addition of trafficking motifs increased BLINK1 presence at the PM up to 30-40 % (cells with measurable current on the total of transfected cells) but the channel lost its light regulation. The best results were obtained with the clone renamed BLINK2, that shows a moderate improvement of expression rate (26% of transfected HEK 293T cells) but intact light regulation of the current. However, BLINK2 has slower kinetics (t1/2 on= 5 min; t1/2 off= 8 min) than the parental channel BLINK1 (on= 87s; off 168 s) and a 60 to 90 s delay in opening after light on. To improve channel kinetics, we have introduced mutations known to tune the LOV domain photocycle: BLINK2 Q513D shows indeed a reduced delay in opening (30 sec). BLINK2 and BLINK2 Q513D are currently under investigation for optogenetic applicability in vivo, both in zebrafish and mouse models.
Improving trafficking and kinetics of a synthetic light-gated Potassium channel / L. S. Alberio, G. Defranceschi, F. Simeoni, P. Zuccolini, G. Thiel, A. Moroni. ((Intervento presentato al 61. convegno Biophysical Society tenutosi a New Orleans nel 2017.
|Titolo:||Improving trafficking and kinetics of a synthetic light-gated Potassium channel|
ALBERIO, LAURA SILVIA (Primo)
|Data di pubblicazione:||feb-2017|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||Improving trafficking and kinetics of a synthetic light-gated Potassium channel / L. S. Alberio, G. Defranceschi, F. Simeoni, P. Zuccolini, G. Thiel, A. Moroni. ((Intervento presentato al 61. convegno Biophysical Society tenutosi a New Orleans nel 2017.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|