The importance of the potassium ion (K+) is coupled to the sodium ion (Na+), because the cell expends a major part of its metabolic energy maintaining the concentrations of Na+ and K+ within the cell. Intracellular concentration ranges are 10-40 mM for Na+ and 120-400 mM for K+. Extracellular concentration ranges are 4-40 mM for K+ and 120-400 mM for Na+.
In the absence of a K+ indicator, efforts have been directed to using indirect techniques to measure or detect K+, including:
- Analogs like thallium or cesium to monitor K+ fluxes
- The pH indicator BCECF AM and the ionophore nigericin in flow cytometry studies
- Combinations of ion selective carriers
- Ion-channel mediated K+ fluxes with membrane potential changes registered by voltage sensitive dyes
- Fiber-optic sensors for K+ with pH sensitive dyes
IThese alternate techniques were necessary because the previously reported fluorescent potassium indicator PBFI requires UV excitation and suffers from poor loading and poor brightness. Ion Potassium Green 2 (IPG-2) successfully addresses these problems. The properties of the K+ indicators are shown in Table 3.1.
Table 3.1. Properties of Potassium Indicators
|IPG-2 TMA+ salt||IPG-2 AM||PBFI K+ salt||PBFI AM|
|Excitation (nm)||488 to 517b||488 to 517d||
a The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. These Kd's were measured in simple aqueous buffers as a guideline to the scientist, who should then calibrate the dye to his/her system.
b The excitation maximum is 517 nm; 488nm excitation gives 40% of the maximum.
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 titration to determine Kd for PBFI was performed in 100 mM TMACI, 10mM MOPS with the addition of potassium chloride. No corrections were made for increasing ionic strength or for quenching by chloride.