Fluorescent dyes as a reliable tool in P2X7 receptor-associated pore studies

Abstract

Since the nineteenth century, a great amount of different biological structures and processes have been assessed by fluorescent dyes. Along with the uses of these compounds as vital and histological dyes, some fluorescent dyes have become valuable tools for the study of the pore phenomenon in plasma membranes. Some ion channels capable of forming large conductance channels, such as P2X7, TRPV1, VDAC-1 and the maxi-anion channels transiently alter the plasma membrane permeability, producing pores, which permit the passage of molecules of up to 1,000 Da. In this review, we discuss the uses of the fluorescent dyes chosen in diverse studies of this topic up to now.

Into rather unexplored terrain—transcellular transport across the blood–brain barrier

“…This purinergic receptor is a trimeric membrane protein complex that opens an integral ion channel when activated by ATP. At micromolar levels of ATP, the P2X 7 channel pore is preferentially permeable to small cations (Na + , K + , Ca 2+ ) but high concentrations of ATP (mM range) further allow permeation of larger cations and anions, including ATP, glutamate and fluorescent reporter dyes with an MW up to 1 kDa (Alves et al, ; Ferreira et al, ). Controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route.…”

Pore-Forming Proteins: Fluorescent Dyes to Study the Channel Functionality and Biophysical Properties

“…Among these assays, the cell death assay, flow cytometry, intracellular calcium quantification, cytokine assay, and cell permeabilization stand out. However, for the best performance of these techniques, fluorescent dyes are used according to their physical-chemical characteristics and molecular mass, serving as a strategy to obtain better results and performance of experimental tests [49]. For additional information about some of the techniques mentioned above, more details involving their respective methodologies can be found in the reviews published below [50][51][52].…”

“…Over the years, the term "fluorescence" was coined by physicist George Gabriel Stokes, in addition to presenting a list of fluorescent substances, confirming new advances in the area. With the advancement of science and the years, the first fluorescent dye was synthesized, fluorescein, an anionic dye produced by German chemist Adolf von Baeyer, a molecule that would be used in several laboratory methods in the future [49,55]. Fluorescent dyes are widely used in various specialties within science, among them; the staining of different structures in tissues and cells stands out.…”

“…A vast number of laboratory techniques of molecular and cellular biology in broad fields of research use these molecules, based on their chemical and physical properties according to the method worked, thus being an excellent resource in the investigation and visualization of the topic addressed [56,57]. Among many techniques, the opening function of the high conductance pores mediated through ion channels can be evaluated in experimental tests for the capture of intracellular dye [4,49]. Many dyes are impermeable to the membrane due to their molecular weight and physical characteristics such as positive charge (cationic dyes) and negative charge (anionic dyes).…”

“…As seen previously, mainly in cationic dyes, the charges vary between the charge of 1 + and 2 + , a fact little investigated, and the search for tests in dyes with more tri or tetravalent charges is relevant in the evaluation of these pores. However, pores similar to dyes also have charges, suggesting varied possibilities for molecular interaction of pores with the physicochemical properties of fluorophores, a fact that is not thoroughly investigated in scientific researches [49].…”