Date Published: March 11, 2019
Publisher: Public Library of Science
Author(s): Bryan Tay, Teneale A. Stewart, Felicity M. Davis, Jennifer R. Deuis, Irina Vetter, Randall Lee Rasmusson.
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.
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+ . 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 . Unlike patch-clamp electrophysiology, fluorescence-based assays provide an indirect measure of NaV channel function, with many commercial dyes available that detect changes in membrane potential or intracellular ion concentration instead of directly measuring sodium current.
Here we describe the development of a high-throughput fluorescent no-wash sodium influx assay using the sodium dye indicator ANG-2 and commercially available food dyes as quenchers. While the experimental protocol for ANG-2 outlines the need for a wash step, the addition of commercially available food dyes with ANG-2 negates the need for this step. In our hands, we found the wash step difficult to implement in 384-well format, as HEK293 cells stably expressing NaV1.1–1.8 have low adherence, and often detach after the wash step despite the use of surfaces that improve cell attachment such as CellBIND and Poly-D-lysine. While all of the red-violet food dyes tested effectively quenched background fluorescence of ANG-2, we chose to use Ponceau 4R in a non-analytical standard form (as the food coloring Pillar Box Red (Queen)), as it is inexpensive to purchase and thus economical for use as a quencher in high-throughput assay formats. Interestingly, out of all the food dyes tested, carmine had the poorest signal-to-noise ratio following addition of veratridine. (Fig 4A) This could be a result of carmine being membrane permeable (S2 Fig) and quenching the intracellular ANG-2 fluorescent signal. It should be noted that membrane permeability was not tested for the quenchers described here, and that incubation times beyond 30 min may result in increased levels of intracellular quenching and a loss of signal.