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Application of optogentetic principles for the modulation of the pacemaker current If

Zuccolini, Paolo (2019)
Application of optogentetic principles for the modulation of the pacemaker current If.
Università degli Studi di Milano; Technische Universität Darmstadt
doi: 10.25534/tuprints-00009649
Ph.D. Thesis, Primary publication

Abstract

HCN4 channels control pacemaking of the heart. They are activated by negative voltage and modulated by cAMP. Recently it had been discovered that cyclic di-nucleotides bind to a second site in the channel C-terminus. This counteracts the effect of cAMP on shifting channel activation positive. A bacterial cyclase, which synthesizes c-di-GMP in response to red light, allows engineering of an optogenetic system for remote HCN4 modulation and hence for controlling the heart pace. Two cyclases were used in this work: one constitutively active (Slr1143) and one red ligh-regulated (BphS). Because the latter has some dark activity, it is co-expressed with a phosphodiesterase (YhjH). Recordings of HCN4 activity in HEK293T cells show that Slr1143 affects the voltage dependence of the channel, shifting the activation curve negative with respect to the control. Experiments with BphS show no difference in HCN4 activity between light and dark treated cells. The combined BphS-YhjH expression seemed to be unable to increase the c-di-GMP concentration in a light dependent manner. To examine the effect of light on c-di-GMP production we quantified the cyclic di-nucleotide with an established ELISA assays in HEK293T cells and with an immune-fluorescence method. The latter consisted of monitoring expression of interferon-β in BphS-YhjH expressing T cells. Cyclic di-nucleotides can activate the STING pathway, which augments synthesis of interferon-β. Both methods underlined that Slr1143 and BphS-YhjH system increased the level of c-di-GMP in cells. This activity, however, was not light regulated. The immuno-fluorescence data indicate a slightly higher expression of the constitutive compared to the light-regulated cyclase. This may explain why we observed an effect of the former but not of the latter on HCN4 gating. Eliminating YhjH did not affect the level of c-di-GMP, suggesting that the phosphodiesterase is insufficient for eliminating c-di-GMP dark production. The data confirm previous results in that c-di-GMP is able to modulate HCN4 activity. BphS is not yet suitable as an optogenetic tool because of its high dark activity. This problem may be overcome by increasing the expression/activity of the phosphodiesterase in the next iteration of engineering an optogenetic tool.

Item Type: Ph.D. Thesis
Erschienen: 2019
Creators: Zuccolini, Paolo
Type of entry: Primary publication
Title: Application of optogentetic principles for the modulation of the pacemaker current If
Language: English
Referees: Thiel, Prof. Gerhard ; Moroni, Prof. Anna
Date: 22 March 2019
Place of Publication: Darmstadt
Refereed: 22 March 2019
DOI: 10.25534/tuprints-00009649
URL / URN: https://tuprints.ulb.tu-darmstadt.de/9649
Abstract:

HCN4 channels control pacemaking of the heart. They are activated by negative voltage and modulated by cAMP. Recently it had been discovered that cyclic di-nucleotides bind to a second site in the channel C-terminus. This counteracts the effect of cAMP on shifting channel activation positive. A bacterial cyclase, which synthesizes c-di-GMP in response to red light, allows engineering of an optogenetic system for remote HCN4 modulation and hence for controlling the heart pace. Two cyclases were used in this work: one constitutively active (Slr1143) and one red ligh-regulated (BphS). Because the latter has some dark activity, it is co-expressed with a phosphodiesterase (YhjH). Recordings of HCN4 activity in HEK293T cells show that Slr1143 affects the voltage dependence of the channel, shifting the activation curve negative with respect to the control. Experiments with BphS show no difference in HCN4 activity between light and dark treated cells. The combined BphS-YhjH expression seemed to be unable to increase the c-di-GMP concentration in a light dependent manner. To examine the effect of light on c-di-GMP production we quantified the cyclic di-nucleotide with an established ELISA assays in HEK293T cells and with an immune-fluorescence method. The latter consisted of monitoring expression of interferon-β in BphS-YhjH expressing T cells. Cyclic di-nucleotides can activate the STING pathway, which augments synthesis of interferon-β. Both methods underlined that Slr1143 and BphS-YhjH system increased the level of c-di-GMP in cells. This activity, however, was not light regulated. The immuno-fluorescence data indicate a slightly higher expression of the constitutive compared to the light-regulated cyclase. This may explain why we observed an effect of the former but not of the latter on HCN4 gating. Eliminating YhjH did not affect the level of c-di-GMP, suggesting that the phosphodiesterase is insufficient for eliminating c-di-GMP dark production. The data confirm previous results in that c-di-GMP is able to modulate HCN4 activity. BphS is not yet suitable as an optogenetic tool because of its high dark activity. This problem may be overcome by increasing the expression/activity of the phosphodiesterase in the next iteration of engineering an optogenetic tool.

Alternative Abstract:
Alternative abstract Language

HCN4-Kanäle steuern die Frequenz des Herzschlags. Sie werden durch negative Spannung aktiviert und durch cAMP moduliert. Kürzlich wurde entdeckt, dass auch zyklische Dinukleotide an eine zweite Stelle im C-Terminus des Kanals binden können. Dies wirkt dem Effekt von cAMP auf die Aktivierung des Kanals entgegen. Eine bakterielle Cyclase, die c-di-GMP als Reaktion auf Rotlicht synthetisiert, ermöglicht die Entwicklung eines optogenetischen Systems zur ferngesteuerten HCN4-Modulation und damit zur Steuerung des Herzrhythmus. Zwei Proteine wurden verwendet: eine konstitutiv aktive (Slr1143) und eine rotlichtregulierte (BphS) Cyclase. Da letztere eine gewisse Dunkelaktivität aufweist, wird eine Phosphodiesterase (YhjH) koexprimiert. Aufzeichnungen über die HCN4-Aktivität in HEK293T-Zellen zeigen, dass Slr1143 die Spannungsabhängigkeit von HCN4 beeinflusst; Slr1143 verschiebt die Aktivierungskurve des Kanals relativ zur Kontrolle in Richtung negativer Membranspannungen. Experimente mit BphS zeigen keinen Unterschied in der HCN4-Aktivität zwischen Zellen, die im Hellen oder Dunklen inkubiert wurden. Die kombinierte BphS-YhjH-Expression schien nicht in der Lage zu sein, die c-di-GMP-Konzentration in einer lichtabhängigen Weise zu erhöhen. Um die Wirkung von Licht auf die c-di-GMP-Produktion zu untersuchen, haben wir die zyklische Dinukleotid-Konzentration mit einem etablierten ELISA-Assay in HEK293T-Zellen und mit einer Immunfluoreszenzmethode quantifiziert. Letztere bestand in der Überwachung der Expression von Interferon-β in BphS-YhjH exprimierenden T-Zellen. Zyklische Dinukleotide können den STING-Pfad aktivieren, der die Synthese von Interferon-β verstärkt. Beide Methoden zeigten, dass sowohl Slr1143 als auch das BphS-YhjH-System zu einer Erhöhung des zellulären c-di-GMP-Spiegels führen. Diese Aktivität war jedoch nicht durch Licht reguliert. Die Eliminierung von YhjH hatte keinen Einfluss auf das c-di-GMP-Level, was darauf hindeutet, dass die Phosphodiesterase nicht in der Lage ist, die Produktion von c-di-GMP im Dunklen zu unterbinden. Die Daten bestätigen die bisherigen Ergebnisse, dass c-di-GMP in der Lage ist, die HCN4-Aktivität zu modulieren. BphS ist aufgrund seiner hohen Dunkelaktivität noch nicht als optogenetisches Werkzeug geeignet. Um dieses Problem auf dem Weg zur Konstruktion eines funktionsfähigen optogenetischen Werkzeugs zu überwinden, muss die Expression/Aktivität der Phosphodiesterase erhöht werden.

German
URN: urn:nbn:de:tuda-tuprints-96492
Classification DDC: 500 Science and mathematics > 570 Life sciences, biology
Divisions: 10 Department of Biology
10 Department of Biology > Plant Membrane Biophyscis (20.12.23 renamed in Biology of Algae and Protozoa)
Date Deposited: 15 Dec 2019 20:56
Last Modified: 01 Dec 2023 08:56
PPN:
Referees: Thiel, Prof. Gerhard ; Moroni, Prof. Anna
Refereed / Verteidigung / mdl. Prüfung: 22 March 2019
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