Potassium Indicators

Potassium indicators are molecules that exhibit an increase in fluorescence upon binding K⁺.

Potassium (K) in physiology

Potassium, along with sodium (Na⁺), is one of nature’s most important monovalent metal cations. Ion channels and transporters for K⁺ play a diverse range of functions from modulating neuronal activity to regulating solute balance. Potassium channels are by far the largest and most structurally diverse class of ion channels and much remains unknown about their functions in normal physiology and disease. An increasingly long list of K⁺ channels and K⁺ transporters are being associated with a wide variety of disorders. 

Potassium channel and K⁺ transporter-targeted drugs already provide effective treatments for a wide breadth of indications including epilepsy, autism, type-2 diabetes, and diuresis. Unfortunately, K⁺ channels, particularly the Kv11.1 channel (a.k.a. hERG), may be the unintended target of other therapeutics resulting in sometimes deadly side-effects. As a result, pre-clinical testing of drug candidates to determine their effects on hERG channel activity is required. 

Because expanding our knowledge of K⁺ channels and K⁺ transporters is of such intense interest to both basic and drug discovery researchers, a facile means to measure their activity is critical. Unfortunately, measuring intracellular and extracellular K⁺ levels and dynamics using fluorescent indicators is challenging. In most cells at rest, cytoplasmic K⁺ concentrations are above 100 mM while Na⁺ concentrations are in the 3 – 5 mM range. Conversely, extracellular K⁺ concentrations are typically in the 3-5 mM range while Na⁺ concentrations are above 100 mM. Therefore, an effective K⁺sensor needs to be able to discriminate between K⁺, Na⁺ and other common physiological cations while possessing a K⁺ affinity appropriate to detect physiologically relevant changes in K⁺ concentrations.

 

K⁺-sensitive fluorescent indicators

Extensive research has afforded the ION Potassium Green (IPG) family of excellent K⁺ sensors, formerly known as Asante Potassium Green (APG). These molecules are small, synthetic fluorochromes which incorporate a K⁺-binding moiety. Under conditions where K⁺ is not bound, the fluorescence of the sensor is significantly quenched. When K⁺ is bound, the quenching is relieved, and the fluorescence of the sensor dramatically increases. 

The IPG family of indicators are the best-in-class replacements for the legacy K⁺ indicator, PBFI. Unlike PBFI, the spectral properties of the IPG family of K⁺ indicators make them convenient to use with common filter sets (e.g. YFP and FITC) as well as multiphoton approaches, and the range of K⁺ affinities make them useful for applications as diverse as extracellular K⁺ sensing to monitoring intracellular K⁺ dynamics. The IPG family of K⁺ indicators is compatible with a wide variety of detectors including fluorescent microscopes, plate readers, flow cytometers, and fluorescent indicator-doped solid-state sensors.

 

(EX 525 nm, EM 545 nm, Kd 50 mM) is the lowest affinity of the ION Potassium Green indicators. It is the indicator that is best positioned to respond to modest changes in intracellular K⁺ concentrations resulting from activation of plasma membrane K⁺ channels.

(EX 525 nm, EM 545 nm, Kd 7 mM) has the highest K⁺ affinity of our single-wavelength indicators making it appropriate for conditions where there may be large changes in intracellular K⁺ (e.g. highly active synapses) or as an extracellular K⁺ sensor.

(EX 525 nm, EM 545 nm, Kd 18 mM) is the ION Potassium Green indicator with intermediate affinity. Currently, IPG-2 is our most popular intracellular K⁺ indicator.

Thallium (Tl⁺)-sensitive fluorescent indicators

While the IPG family of dyes are excellent fluorescent K⁺ indicators, the challenges of discriminating between K⁺ and Na⁺ are not trivial, and together with the often modest changes in intracellular or extracellular K⁺ results in relatively small signals compared to those observed with the Ca²-sensitive fluorescent indicators, such as Fluo-4. In many instances, the Tl+ flux assay offers an outstanding alternative to K⁺-sensitive fluorescent indicators. The Tl⁺ flux assay takes advantage of a Tl⁺-selective fluorescent indicator, Thallos, and the fact that K⁺ channels and K⁺ transporters readily accept Tl as a K⁺ surrogate. When Tl is added to the outside of cells loaded with Thallos, Tl entering cells through K channels and K transporters results a dramatic increase in fluorescence. Today, the Tl assay is the most commonly used approach for large-scale high-throughput screening (HTS) of K channels and K transporters.

(EX 490 nm, EM 515 nm) is the best-in-class Tl⁺-sensitive fluorescent indicator. It offers outstanding cell loading and excellent compatibility with commonly available FITC filters.

Indicator forms and formats 

We provide our IPG family of K⁺-sensitive fluorescent indicators in membrane permeable and membrane impermeable forms. We offer Thallos as part of our convenient Brilliant Thallium Assay kits and, unlike other vendors, as a stand-alone product for investigators who prefer to use their own solutions and reagents.

Cell permeable, acetoxymethyl group (AM) esters

The native forms of K⁺-sensitive and Tl⁺-sensitive fluorescent indicators are negatively charged and membrane impermeable. However, masking the negative charge using non-polar, ester-linked moieties (AM esters) allow the molecules to enter cells through passive diffusion. Once inside the cell, ubiquitous intracellular esterase enzymes promote rapid hydrolysis of the AM esters leaving the active, highly polar form of the indicator trapped inside the cell.

Reagents for improving indicator loading and retention

Although the AM forms of ION’s fluorescent K⁺ and Tl⁺ indicators readily cross the plasma membrane, there are other factors that can affect how well the indicators load into cells. Because AM forms of the indicators are quite hydrophobic, it is common to use the non-ionic surfactant, Pluronic F-127, to improve their solubility in aqueous solution thus improving their loading into cells. Pluronic F-127 at the concentrations employed for loading indicators into cells is generally well tolerated. While Pluronic F-127 isn’t absolutely essential for loading most fluorescent K⁺ and Tl⁺ indicators into cells, it is highly recommended. 

Another factor that can affect cell loading and intracellular localization is the presence of transport proteins capable of transporting many ion-sensitive, fluorescent indicators. In many cell types, the activity of these transporters can result in compartmentalization of the indicators into intracellular compartments as well as extrusion of the indicators outside of the cell. Both of these processes can dramatically reduce the signal-to-background levels and in some cell types (e.g. CHO cells),  make adequate loading of K⁺-selective fluorescent indicators nearly impossible. Fortunately, inhibitors of these transport proteins can be an effective means of improving cytoplasmic localization and indicator retention. ION offers a convenient, 100X ION-Pro inhibitor solution that may be used in conjunction with any of our K⁺-sensitive fluorescent indicators.

Be Brilliant! Explore our line of Potassium Indicators

Our collection of membrane permeable and impermeable ion indicators targeting K⁺.

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High-throughput, no wash thallium (Tl⁺) assay. An optimal solution for measuring flux through potassium (K⁺), sodium (Na⁺), and non-selective cation channels and their effectors including transporters and GPCRs. 100 plates

490/515
FITC, GFP
$3,270.00

High-throughput, no wash thallium (Tl⁺) assay. An optimal solution for measuring flux through potassium (K⁺), sodium (Na⁺), and non-selective cation channels and their effectors including transporters and GPCRs. 10 plates

490/515
FITC, GFP
$430.00

Yellow-green fluorescent, potassium (K⁺) indicator, membrane permeable. Lower K⁺ affinity than IPG-2 and IPG-4.

525/545
50
DMSO
GFP, FITC, YFP
$340.00

Yellow-green fluorescent, potassium (K⁺) indicator, membrane impermeable. Lower K⁺ affinity than IPG-2 and IPG-4.

525/545
50
H2O
GFP, FITC, YFP
$290.00

Yellow-green fluorescent, potassium (K⁺) indicator, membrane permeable. Lower K⁺ affinity than IPG-4 and higher affinity than IPG-1

525/545
18
DMSO
GFP, FITC, YFP
$340.00

Yellow-green fluorescent, potassium (K⁺) indicator, membrane impermeable. Lower K+ affinity than IPG-4 and higher affinity than IPG-1

525/545
18
H2O
GFP, FITC, YFP
$290.00

Yellow-green fluorescent, potassium (K⁺) indicator, membrane permeable. Higher K⁺ affinity than IPG-1 and IPG-2

525/545
7
DMSO
GFP, FITC, YFP
$340.00

Yellow-green fluorescent, potassium (K⁺) indicator, membrane impermeable. Higher K⁺ affinity than IPG-1 and IPG-2

525/545
7
H2O
GFP, FITC, YFP
$290.00

Best in class, green fluorescent, thallium (Tl⁺) indicator, membrane permeable. Works with potassium (K⁺), sodium (Na⁺), and monovalent cation channels, and transporters.

490/515
DMSO
FITC, GFP
$495.00
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