Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels.

Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling

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

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

Engineering of a light-gated potassium channel

The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K+) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light–induced K+ channel 1 (BLINK1) engineered by fusing the plant LOV2-Ja photosensory module to the small viral K+ channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K+ selectivity and high single-channel conductance, but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K+ equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities

Characterization of an N-terminal Na v 1.5 channel variant - a potential risk factor for arrhythmias and sudden death?

BACKGROUND Alterations in the SCN5A gene encoding the cardiac sodium channel Na1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. METHODS Mutant as well as wild type GFP tagged Na1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. RESULTS Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Na1.5 current over the entire voltage window. CONCLUSION The results support the assumption that the detected sequence aberration alters Na1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death

Optogenetics. Engineering of a light-gated potassium channel.

The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities