Abstract
Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1–1.7 activator veratridine (EC50 10–29 μM) or the NaV1.1–1.8 inhibitor tetracaine (IC50’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.
Highlights
Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias
All of the red and violet food dyes tested, including Brilliant Black BN, Carmine, Ponceau 4R, Allura Red and Amaranth, absorbed at wavelengths that overlapped with the peak emission of Asante NaTRIUM Green-2 (ANG-2) (540 nm) and the FLIPR detection range (515– 575 nm) (Fig 2B), with varying peak absorbances and ranges (Fig 2C)
We hypothesized that these dyes could be used to quench background fluorescence in cells loaded with ANG-2
Summary
Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. The NaV subfamily consists of nine α subunits (NaV1.1–1.9), which are responsible for the generation and propagation of action potentials in neurons. The ~260kDa proteins form an ion-selective pore which opens upon membrane depolarization to allow influx of Na+ [1]. While patch-clamp electrophysiology remains the gold standard for assessing NaV channel function, fluorescence-based assays are routinely used to screen vast chemical libraries for novel drug leads, as these assays are comparatively cheap, high-throughput and less technically challenging to perform [2]. Unlike patch-clamp electrophysiology, fluorescence-based assays provide an indirect measure of NaV channel function, with many.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
