Molecular Biology


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0 Q&A 706 Views Aug 20, 2024

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes—expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons.

0 Q&A 7872 Views Oct 5, 2018
The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses in vitro is problematic and expensive and, accordingly, only limited information on the purified channels is available in the literature. Here we describe a protocol for binding studies of fluorescently labeled cyclic nucleotides to a homologue of eukaryotic CNG channels. Furthermore, we describe how to directly probe binding of unlabeled cyclic nucleotides in a competition assay. The use of fluorescence as a sensitive probe for ligand binding reduces the amount of protein needed and enables fast and easy measurements using standard laboratory equipment.
0 Q&A 9196 Views Apr 20, 2017
Mechanically-gated ion channels play key roles in mechanotransduction, a process that translates physical forces into biological signals. Epithelial and endothelial cells are exposed to laminar shear stress (LSS), a tangential force exerted by flowing fluids against the wall of vessels and epithelia. The protocol outlined herein has been used to examine the response of ion channels expressed in Xenopus oocytes to LSS (Hoger et al., 2002; Carattino et al., 2004; Shi et al., 2006). The Xenopus oocyte is a reliable system that allows for the expression and chemical modification of ion channels and regulatory proteins (George et al., 1989; Palmer et al., 1990; Sheng et al., 2001; Carattino et al., 2003). Therefore, this technique is suitable for studying the molecular mechanisms that allow flow-activated channels to respond to LSS.



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