Related Technical Notes
Sodium and potassium channels are ion-selective protein pores that span the cell's plasma membrane and serve to establish and regulate membrane potential. They are typically classified according to their response mechanism: voltage-gated channels open or close in response to changes in membrane potential, whereas ligand-gated or ion-activated channels are triggered by ligand or ion binding. In excitable cells such as neurons and myocytes, these channels function both to create the action potential and to reset the cell's resting membrane potential.
In this section, we describe sodium- and potassium-selective fluorescent indicators, as well as the FluxOR Thallium Detection Kits, which provide a fluorescence-based method for assaying potassium ion channel and transporter activities. The next section describes fluorescent indicators for intracellular and extracellular chloride, together with an assortment of analytical reagents and methods for direct or indirect quantitation of other inorganic anions (Detecting Chloride, Phosphate, Nitrate and Other Anions—Section 21.2).
Properties of SBFI and PBFI
SBFI and PBFI are fluorescent indicators for sodium and potassium, respectively. Although the selectivity of SBFI and PBFI for their target ions is less than that of calcium indicators such as fura-2, it is sufficient for the detection of physiological concentrations of Na+ and K+ in the presence of other monovalent cations. Furthermore, the spectral responses of SBFI and PBFI upon ion binding permit excitation ratio measurements (Loading and Calibration of Intracellular Ion Indicators—Note 19.1), and these indicators can be used with the same optical filters and equipment used for fura-2.
SBFI () and PBFI () comprise benzofuranyl fluorophores linked to a crown ether chelator. The cavity size of the crown ether confers selectivity for Na+ versus K+ (or vice versa in the case of PBFI). When an ion binds to SBFI or PBFI, the indicator's fluorescence quantum yield increases, its excitation peak narrows and its excitation maximum shifts to shorter wavelengths (), causing a significant change in the ratio of fluorescence intensities excited at 340/380 nm (Figure 21.1.1, Figure 21.1.2). This fluorescence signal is slightly sensitive to changes in pH between 6.5 and 7.5, but it is strongly affected by ionic strength and viscosity. Researchers have described the use of SBFI for emission ratio detection (410/590 nm, excited at 340 nm). More recently, the implementation of two-photon excitation of SBFI with infrared light has been reported for Na+ imaging in spines and fine dendrites of central neurons ().
Although SBFI is quite selective for the Na+ ion, K+ has some effect on the native affinity of SBFI for Na+ (Figure 21.1.1). The dissociation constant (Kd) of SBFI for Na+ is 3.8 mM in the absence of K+, and 11.3 mM in solutions with a combined Na+ and K+ concentration of 135 mM, which approximates physiological ionic strength. SBFI is ~18-fold more selective for Na+ than for K+. Likewise, the Kd of PBFI for K+ is strongly dependent on whether Na+ is present (Figure 21.1.2), with a value of 5.1 mM in the absence of Na+ and 44 mM in solutions with a combined Na+ and K+ concentration of 135 mM. In buffers in which the Na+ is replaced by tetramethylammonium chloride, the Kd of PBFI for K+ is 11 mM; choline chloride and N-methylglucamine are two other possible replacements for Na+ in the medium. Although PBFI is only 1.5-fold more selective for K+ than for Na+, this selectivity is often sufficient because intracellular K+ concentrations are normally about 10 times higher than Na+ concentrations.
The Kd of all ion indicators depends on factors such as pH, temperature, ionic strength, concentrations of other ions and dye–protein interactions. Due to these environmental factors, the Kd determined in situ for intracellular SBFI is substantially higher than that determined in cell-free buffer solutions. Kd (Na+) values of 29 mM, 26.6 mM and 18.0 mM have been determined for SBFI in lizard peripheral axons, porcine adrenal chromaffin cells and rat hippocampal neurons, respectively. Consequently, intracellular SBFI should be calibrated using the pore-forming antibiotic gramicidin (G6888). Palytoxin, an ionophoric toxin isolated from marine coelenterates, is much more effective than gramicidin for equilibrating intracellular and extracellular Na+. Intracellular PBFI should be calibrated using the K+ ionophore valinomycin (V1644).
|Figure 21.1.1 The excitation spectral response of SBFI (S1262) to Na+: A) in K+-free solution and B) in solutions containing K+ with the combined Na+ and K+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.||Figure 21.1.2 The excitation spectral response of PBFI (P1265MP) to K+: A) in Na+-free solution and B) in solutions containing Na+ with the combined K+ and Na+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.|
Cell Loading with SBFI and PBFI
SBFI and PBFI are available both as cell-impermeant acid salts (S1262, P1265MP) and as cell-permeant acetoxymethyl (AM) esters (S1263, S1264, P1267MP). The anionic acid forms can be loaded into cells using our Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), or by microinjection, patch-pipette infusion or electroporation. For AM ester loading (Loading and Calibration of Intracellular Ion Indicators—Note 19.1), addition of the Pluronic F-127 (P3000MP, P6866, P6867) or PowerLoad (P10020) dispersing agents as well as relatively long incubation times—up to four hours—are typically necessary. ATP-induced permeabilization reportedly produces increased uptake of SBFI AM by bovine pulmonary arterial endothelial cells (BPAEC). Somewhat higher working concentrations of PBFI and SBFI than those used for fura-2 may be required because of the lower fluorescence quantum yields of these indicators. AM ester loading sometimes produces intracellular compartmentalization of SBFI. As with other AM esters, reducing the incubation temperature below 37°C may inhibit compartmentalization. Other practical aspects of loading and calibrating SBFI have been reviewed by Negulescu and Machen.
Applications of SBFI
Applications of PBFI
PBFI has fewer documented applications than SBFI. Renewed interest has been prompted by the observation that intracellular K+ levels appear to be a controlling factor in apoptotic cell death pathways. Flow cytometric measurements using UV argon-ion laser excitation (351 nm and 364 nm) of PBFI indicate that K+ efflux induces shrinkage of apoptotic cells and is a trigger for caspase activation. Furthermore, PBFI provides a potential alternative to radiometric K+ efflux assays using 86Rb. Other applications of PBFI include:
- Detecting adrenoceptor-stimulated decreases of intracellular K+ concentration in astrocytes and neurons
- Evaluating the mediating effects of K+ depletion on monocytic cell necrosis
- Investigating the relationship between cytoplasmic K+ concentrations and NMDA excitotoxicity
- Measuring intracellular K+ fluxes associated with apoptotic cell shrinkage
- Monitoring mitochondrial KATP channel activation
- Quantitating K+ in isolated cochlear outer hair cells and in mammalian ventricles using patch-clamp techniques
- Detecting elevated intracellular K+ levels associated with HIV-induced cytopathology
- Measuring K+ levels in plant cells and vacuoles
The Sodium Green indicator can be excited at 488 nm (), providing a valuable alternative to the UV light–excitable SBFI for use with confocal laser-scanning microscopes and flow cytometers. We offer the cell-impermeant tetra(tetramethylammonium) salt of the Sodium Green indicator (S6900), as well as its cell-permeant tetraacetate (S6901).
The Sodium Green indicator comprises two 2',7'-dichlorofluorescein dyes linked to the nitrogen atoms of a crown ether () with a cavity size that confers selectivity for the Na+ ion. Upon binding Na+, the Sodium Green indicator exhibits an increase in fluorescence emission intensity with little shift in wavelength (Figure 21.1.3). Although the Sodium Green indicator lacks the direct ratiometric readout capability of SBFI, fluorescence intensity fluctuations due to cell size variability can be compensated to some extent by using forward light scatter as a reference signal in flow cytometry.
As compared with SBFI, the Sodium Green indicator shows greater selectivity for Na+ than K+ (~41-fold versus ~18-fold) and displays a much higher fluorescence quantum yield (0.2 versus 0.08) in Na+-containing solutions. The longer-wavelength absorption of the Sodium Green indicator results in reduction of the potential for photodamage to the cell because the energy of the excitation light is lower than that of the UV light required for excitation of SBFI. The Kd of the Sodium Green indicator for Na+ is about 6 mM in K+-free solution and about 21 mM in solutions with combined Na+ and K+ concentration of 135 mM, approximating physiological ionic strength. Because its Kd may be shifted due to intracellular interactions, the Sodium Green indicator should be calibrated in situ using the pore-forming antibiotic gramicidin (G6888). In some cases, dye–protein interactions may cause severe dampening or even complete elimination of the Na+-dependent fluorescence response of intracellular Sodium Green indicator. Nevertheless, flow cytometric measurements in Chinese hamster ovary (CHO) are well correlated with spectrofluorometric measurements using SBFI. Other applications include:
- Assessing the regulation of Na+/K+-ATPase by persistent Na+ accumulation in rat thalamic neurons
- Confocal imaging of Na+ transport in rat colonic mucosa and cochlear hair cells by flow cytometry
- Detecting anoxia-induced Na+ influx in neurons
- Determining intracellular Na+ concentration in crayfish presynaptic terminals using an area-ratio method
- Fluorescence lifetime imaging of intracellular Na+
- Measuring intracellular Na+ concentration in bacterial cells and green algae
- Determining voltage-gated sodium channel NaV1.5–driven endosomal Na+ levels in macrophages
Figure 21.1.3 Emission spectral response of the Sodium Green indicator (S6900) to Na+: A) in K+-free solution and B) in solutions containing K+ with the combined Na+ and K+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.
CoroNa Green Na+ Indicator
The CoroNa Green dye is a green-fluorescent Na+ indicator that exhibits an increase in fluorescence emission intensity upon binding Na+ (excitation/emission = 492/516 nm), with little shift in wavelength (Figure 21.1.4). Similar to our SBFI and Sodium Green Na+ indicators, the CoroNa Green indicator allows spatial and temporal resolution of Na+ concentrations in the presence of physiological concentrations of other monovalent cations. CoroNa Green Na+ indicator has been co-loaded with Alexa Fluor 594 dextran (an ion-insensitive reference) via suction pipettes into live rat optic nerves for confocal imaging of intracellular Na+ levels; calcium measurements were also made using fluo-4 dextran and Alexa Fluor 594 dextran.
Comprising a fluorescein molecule linked to a crown ether with a cavity size that confers selectivity for the Na+ ion (), the CoroNa Green indicator is less than half the size of the Sodium Green indicator (molecular weight 586 and 1668, respectively). This smaller size appears to help the cell-permeant CoroNa Green AM load cells more effectively than the Sodium Green tetraacetate. Furthermore, the CoroNa Green indicator responds to a broader range of Na+ concentration, with a Kd of ~80 mM. The cell-impermeant CoroNa Green indicator (C36675) is supplied in a unit size of 1 mg. The cell-permeant AM ester of the CoroNa Green indicator (C36676) is supplied as a set of 20 vials, each containing 50 µg of the indicator.
Figure 21.1.4 Fluorescence emission spectra of the CoroNa Green indicator (C36675, C36676) in 50 mM MOPS, pH 7.0 (adjusted with tetramethylammonium hydroxide), containing 100 mM K+ and variable concentrations of Na+ as indicated.
CoroNa Red Na+ Indicator
CoroNa Red chloride is based on a crown ether that has structural similarity to the Ca2+ chelator BAPTA (). Unlike SBFI and the Sodium Green indicator, the net positive charge of CoroNa Red chloride targets the indicator to mitochondria (), and therefore loading of cells does not require use of a permeant ester derivative of the dye. Cells are typically loaded by adding 0.5–1.0 µM CoroNa Red chloride from a 1 mM stock solution in DMSO, incubating for 10–30 minutes at 37°C and finally washing with dye-free medium before commencing fluorescence analysis. The CoroNa Red indicator is only weakly fluorescent in the absence of Na+ and its fluorescence increases ~15-fold upon binding Na+ (Figure 21.1.5). Despite its relatively high Kd for Na+ of ~200 mM, the CoroNa Red indicator exhibits sensitive responses to cellular Na+ influxes through voltage-gated channels and ATP-gated cation pores. Verkman and co-workers have immobilized the CoroNa Red indicator on polystyrene microspheres and used this complex to measure Na+ concentrations around 100 mM in the tracheal airway–surface liquid (ASL) of cultured epithelial cells and human lung tissues. The CoroNa Red indicator has also been employed to investigate the Na+ channel permeation pathway using polyhistidine-tagged and pore-only constructs of a voltage-dependent Na+ channel. The CoroNa Red indicator is available as a single 1 mg vial (C24430) or as a set of 20 vials, each containing 50 µg of the indicator (C24431).
Figure 21.1.5 Fluorescence emission spectra of the CoroNa Red indicator (C24430, C24431) in 50 mM MOPS (pH 7.0, adjusted with tetramethylammonium hydroxide) containing 100 mM K+ and variable concentrations of Na+ as indicated.
Assaying K+ Channels with the FluxOR Potassium Ion Channel Assay Kit
The FluxOR Potassium Ion Channel Assay Kits (F10016, F10017) provide a fluorescence-based assay for high-throughput screening (HTS) of potassium ion channel and transporter activities. The FluxOR Potassium Ion Channel Assay Kits take advantage of the well-described permeability of potassium channels to thallium (Tl+) ions. When thallium is present in the extracellular solution containing a stimulus to open potassium channels, channel activity is detected with a cell-permeant thallium indicator dye that reports large increases in fluorescence emission at 525 nm as thallium flows down its concentration gradient and into the cells (Figure 21.1.6). In this way, the fluorescence reported in the FluxOR system becomes a surrogate indicator of activity for any ion channel or transporter that is permeable to thallium, including the human ether-a-go-go–related gene (hERG) channel, one of the human cardiac potassium channels. The FluxOR potassium ion channel assay has been validated for homogeneous high-throughput profiling of hERG channel inhibition using BacMam-mediated transient expression of hERG (see below). The FluxOR Potassium Ion Channel Assay Kits can also be used to study potassium co-transport processes that accommodate the transport of thallium into cells. Furthermore, resting potassium channels and inward rectifier potassium channels like Kir2.1 can be assayed by adding stimulus buffer with thallium alone, without any depolarization to measure the signal.
The FluxOR reagent, a thallium indicator dye, is loaded into cells as a membrane-permeable AM ester. The FluxOR dye is dissolved in DMSO and further diluted with FluxOR assay buffer, a physiological HBSS (Hank's balanced salt solution), for loading into cells. Loading is assisted by the proprietary PowerLoad concentrate, an optimized formulation of nonionic Pluronic surfactant polyols that act to disperse and stabilize AM ester dyes for optimal loading in aqueous solution. This PowerLoad concentrate is also available separately (P10020) to aid the solubilization of water-insoluble dyes and other materials in physiological media.
Once inside the cell, the nonfluorescent AM ester of the FluxOR dye is cleaved by endogenous esterases into a weakly fluorescent (basal fluorescence), thallium-sensitive indicator. The thallium-sensitive form is retained in the cytosol, and its extrusion is inhibited by water-soluble probenecid (P36400, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), which blocks organic anion pumps. For most applications, cells are loaded with the dye at room temperature. For best results, the dye-loading buffer is then replaced with fresh, dye-free assay buffer (composed of physiological HBSS containing probenecid), and cells are ready for the HTS assay.
Each FluxOR Potassium Ion Channel Assay Kit contains:
- FluxOR reagent
- FluxOR assay buffer
- PowerLoad concentrate
- FluxOR chloride-free buffer
- Potassium sulfate (K2SO4) concentrate
- Thallium sulfate (Tl2SO4) concentrate
- Dimethylsulfoxide (DMSO)
- Detailed protocols (FluxOR Potassium Ion Channel Assay)
The FluxOR Kits provide a concentrated thallium solution along with sufficient dye and buffers to perform ~4000 (F10016) or ~40,000 (F10017) assays in a 384-well microplate format. These kits allow maximum target flexibility and ease of operation in a homogeneous format. The FluxOR potassium ion channel assay has been demonstrated for use with CHO and HEK 293 cells stably expressing hERG, as well as U2OS cells transiently transduced with the BacMam hERG reagent (B10019, B10033; see below) (Figure 21.1.7).
Figure 21.1.6 Thallium redistribution in the FluxOR assay. Basal fluorescence from cells loaded with FluxOR reagent (provided in the FluxOR Potassium Ion Channel Assay Kits; F10016, F10017) is low when potassium channels remain unstimulated, as shown in the left panel. When thallium is added to the assay with the stimulus, the thallium flows down its concentration gradient into the cells, activating the dye as shown in the right panel.
Figure 21.1.7 FluxOR potassium ion channel assays (F10016, F10017) performed on fresh and frozen U2OS cells transduced with the BacMam hERG reagent (B10019, B10033). A) Raw data (RFU = relative fluorescence units) obtained in the FluxOR assay determination of thallium flux in U2OS cells transduced with BacMam-hERG and kept frozen until the day of use. The arrow indicates the addition of the thallium/potassium stimulus, and upper and lower traces indicate data taken from the minimum and maximum doses of cisapride used in the determination of the dose-response curves. B) Raw pre-stimulus peak and baseline values were boxcar averaged and normalized, and indicate the fold increase in fluorescence over time. C) Data generated in a dose-response determination of cisapride block on BacMam hERG expressed in U2OS cells freshly prepared from overnight expression after viral transduction. D) Parallel data obtained from cells transduced with BacMam-hERG, stored for 2 weeks in liquid nitrogen, thawed, and plated 4 hours prior to running the assay. Error bars indicate standard deviation, n = 4 per determination.
Using BacMam Technology for Transient Expression of K+ Channels
Potassium channel cDNAs that have been engineered into a baculovirus gene delivery/expression system using BacMam technology (BacMam Gene Delivery and Expression Technology—Note 11.1) are also available for use with the FluxOR Potassium Ion Channel Assay Kits, including the human ether-a-go-go related gene (hERG) (Figure 21.1.8), several members of the voltage-gated K+ channel (Kv) gene family and two members of the inwardly rectifying K+ channel (Kir) gene family:
- BacMam hERG (for 10 microplates, B10019; for 100 microplates, B10033)
- BacMam Kv1.1 (for 10 microplates, B10331)
- BacMam Kv1.3 (for 10 microplates, B10332)
- BacMam Kv2.1 (for 10 microplates, B10333)
- BacMam Kv7.2 and Kv7.3 (for 10 microplates, B10147)
- BacMam Kir1.1 (for 10 microplates, B10334)
- BacMam Kir2.1 (for 10 microplates, B10146)
The BacMam system uses a modified insect cell baculovirus as a vehicle to efficiently deliver and express genes in mammalian cells with minimum effort and toxicity. The use of BacMam delivery in mammalian cells is relatively new, but well described, and has been used extensively in a drug discovery setting. Constitutively expressed ion channels and other cell surface proteins have been shown to contribute to cell toxicity in some systems, and may be subject to clonal drift and other inconsistencies that hamper successful experimentation and screening. Thus, inducible, division-arrested or transient expression systems such as BacMam technology are increasingly methods of choice to decrease variability of expression in such assays.
U2OS cells (ATCC number HTB-96) have been shown to demonstrate highly efficient expression of BacMam delivered targets in a null background ideal for screening in a heterologous expression system. The U2OS cell line is recommended for use if your particular cell line does not efficiently express the BacMam targets. Examples of other cell lines that are efficiently transduced by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.
Figure 21.1.8 BacMam-hERG gene delivery and expression. This schematic depicts the mechanism of BacMam-mediated gene delivery into a mammalian cell and expression of the hERG gene (B10019, B10033). The hERG gene resides within the baculoviral DNA, downstream of a CMV promoter that drives its expression when introduced into a mammalian target cell. BacMam viral particles are taken up by endocytic pathways into the cell, and the DNA within them is released for transcription and expression. The translated protein is then folded for insertion into the membrane, forming functional hERG ion channels. This process begins within 4–6 hours and in many cell types is completed after an overnight period.
|Low Ion *||High Ion *|
|C24430||773.32||L||DMSO||547||92,000||570||H2O||551||92,000||576||H2O/Na+||200 mM||1, 2, 3, 4|
|C24431||773.32||L||DMSO||547||92,000||570||H2O||551||92,000||576||H2O/Na+||200 mM||1, 2, 3, 4|
|C36675||585.56||F,D,L||pH >6||492||68,000||516||H2O||492||68,000||516||H2O/Na+||80 mM||1, 2, 5, 6|
|P1265MP||950.99||L||pH >6||336||33,000||557||H2O||338||41,000||507||H2O/K+||5.1 mM||1, 5, 7|
|S1262||906.94||L||pH >8||339||45,000||565||H2O||333||52,000||539||H2O/Na+||3.8 mM||1, 5, 9|
|S6900||1667.57||L||pH >6||506||117,000||532||H2O||507||133,000||532||H2O/Na+||6.0 mM||1, 2, 5, 9|
* For "Low Ion" spectra, the concentration of Na+ or K+ is zero. For "High Ion" spectra, the concentration of Na+ or K+ is in excess of that required to saturate the response of the indicator.