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.

STRATEGIES FOR AN OPTOGENETIC MODULATION OF THE PACEMAKER CURRENT IF / P. Zuccolini ; tutors: A. Moroni A. ; scientific committee: Thiel G., Schroeder I., Barbuti A. ; reviewers: Hansen U., Schroeder I. - : . DIPARTIMENTO DI BIOSCIENZE, 2019 Mar 22. ((31. ciclo, Anno Accademico 2018. [10.13130/zuccolini-paolo_phd2019-03-22].

STRATEGIES FOR AN OPTOGENETIC MODULATION OF THE PACEMAKER CURRENT IF

P. Zuccolini
2019-03-22

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.
MORONI, ANNA
MORONI, ANNA
Settore BIO/11 - Biologia Molecolare
STRATEGIES FOR AN OPTOGENETIC MODULATION OF THE PACEMAKER CURRENT IF / P. Zuccolini ; tutors: A. Moroni A. ; scientific committee: Thiel G., Schroeder I., Barbuti A. ; reviewers: Hansen U., Schroeder I. - : . DIPARTIMENTO DI BIOSCIENZE, 2019 Mar 22. ((31. ciclo, Anno Accademico 2018. [10.13130/zuccolini-paolo_phd2019-03-22].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/634438
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