GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers

Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP-MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3.
Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP-MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP-MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP-MgTx as a component of an analytical system based on the hybrid KcsA-Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP-MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.

Nanobody-Based GFP Traps to Study Protein Localization and Function in Developmental Biology

Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies to well-characterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms.

Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells.
The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.

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