Fluorescent Sodium Indicators
There is a large difference in the sodium ion (Na+) concentration inside and outside the cell (5-40 mM intracellular; 120-450 mM extracellular, depending on organism). This concentration gradient is used to power nutrient uptake, to regulate concentrations of other intracellular ions and solutes, and to generate and transmit electrical impulses in excitable cells such as nerve and muscle. These functions are so important that organisms devote a major part of their metabolic energy to maintaining the sodium gradient. The low intracellular Na+ concentration requires that a Na+ indicator have the sensitivity to measure any small changes that occur. Moreover, intracellular potassium ion (K+) concentration is typically high (in excess of 100 mM); therefore a Na+ indicator should respond selectively to Na+, not K+.
Table 2.1 lists the properties of sodium indicators offered by TEFLabs.
b The titration to determine Kd for SBFI was performed in 100 mM TMACl, 10 mM MOPS with the addition of sodium chloride. No corrections were made for increasing ionic strength or quenching by chloride.
c The titrations to determine Kd were performed in 140 mM TMACl, 10 mM MOPS, pH 7.1 with the addition of sodium chloride. No corrections were made for increasing ionic strength or quenching by chloride.
d Once the AM ester form permeates the cell membrane, non-specific esterases hydrolyze the AM esters, taking the indicator to its active salt form.
e The excitation maximum is 517 nm; 488 nm excitation gives 40% of the maximum.
Two sets of dyes are represented in Table 2.1: SBFI and the two forms of ANG.
SBFI, introduced in 1989, has been the dye most frequently used for measuring Na+ until now. Unfortunately, SBFI requires excitation by UV light and is difficult to load. Previous efforts to replace SBFI by visible wavelength indicators have yielded Sodium GreenTM, CoroNaTM Red, and CoroNaTM Green, but they have problems associated with ineffectiveness in the cell, inability to detect small Na+ changes, and leakage from cells. (Sodium GreenTM, and CoroNaTM are trademarks of Molecular Probes division of Life Technologies.)
Our new Na+ indicators, ANG-1 (Asante NaTRIUM GreenTM 1) and ANG-2 ( Asante NaTRIUM GreenTM 2), display a range of desirable characteristics, including high sensitivity, high loading efficiency, and excellent brightness and dynamic range. These indicators are excited by visible light and thus permit sodium studies to be conducted on equipment already in use for calcium high throughput screening and for cell and ion channel studies.
Table 2.2 compares SBFI’s performance with Asante NaTRIUM Green.