Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome.

Some missense gain-of-function mutations in CACNA1C gene, encoding calcium channel CaV1.2, cause a life-threatening form of long QT syndrome named Timothy syndrome, with currently no clinically-effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in iPSC-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of long QT syndrome, type 1 (LQTS1) and 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that an FDA-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1, to shorten the prolonged action potential in Timothy syndrome cardiomyocytes and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in Timothy syndrome model mice. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.

GEM-IL: A highly responsive fluorescent lactate indicator.

Lactate metabolism has been shown to have increasingly important implications in cellular functions as well as in the development and pathophysiology of disease. The various roles as a signaling molecule and metabolite have led to interest in establishing a new method to detect lactate changes in live cells. Here we report our development of a genetically encoded metabolic indicator specifically for probing lactate (GEM-IL) based on superfolder fluorescent proteins and mutagenesis. With improvements in its design, specificity, and sensitivity, GEM-IL allows new applications compared with the previous lactate indicators, Laconic and Green Lindoblum. We demonstrate the functionality of GEM-IL to detect differences in lactate changes in human oncogenic neural progenitor cells and mouse primary ventricular myocytes. The development and application of GEM-IL show promise for enhancing our understanding of lactate dynamics and roles.

Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP.

Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent noncanonical amino acid 3-((6-acetylnaphthalen-2-yl)amino)-2-aminopropanoic acid (ANAP) into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells, and they exhibited pathophysiological function. A spectral imaging potassium depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells.

Optimized light-inducible transcription in mammalian cells using Flavin Kelch-repeat F-box1/GIGANTEA and CRY2/CIB1.

Light-inducible systems allow spatiotemporal control of a variety of biological activities. Here, we report newly optimized optogenetic tools to induce transcription with light in mammalian cells, using the Arabidopsis photoreceptor Flavin Kelch-repeat F-box 1 (FKF1) and its binding partner GIGANTEA (GI) as well as CRY2/CIB1. By combining the mutagenesis of FKF1 with the optimization of a split FKF1/GI dimerized Gal4-VP16 transcriptional system, we identified constructs enabling significantly improved light-triggered transcriptional induction. In addition, we have improved the CRY2/CIB1-based light-inducible transcription with split construct optimization. The improvements regarding the FKF1/GI- and CRY2/CIB1-based systems will be widely applicable for the light-dependent control of transcription in mammalian cells.