Probes for Ion Channels and Carriers—Section 16.3

Page Contents

• Probes for Ca2+ Channels and Carriers
• Probes for Na+ Channels and Carriers
• Probes for K+ Channels and Carriers
• Probes for Anion Transporters
• Data Table
• Ordering Information

This section describes a variety of probes for Ca²⁺, Na⁺, K⁺ and Cl^– ion channels and carriers. Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19, pH Indicators—Chapter 20 and Indicators for Na+, K+, Cl– and Miscellaneous Ions—Chapter 21 contain our extensive selection of indicators for these physiologically important ions, providing a means of correlating ion channel activation with subsequent changes in intracellular ion concentration. Ion flux also affects the cell's membrane potential, which can be measured with the probes described in Probes for Membrane Potential—Chapter 22.

In both excitable and nonexcitable cells, intracellular Ca²⁺ levels modulate a multitude of vital cellular processes—including gene expression, cell viability, cell proliferation, cell motility and cell shape and volume regulation—and thereby play a key role in regulating cell responses to external activating agents. These dynamic changes in intracellular Ca²⁺ levels are regulated by ligand-gated and G-protein–coupled ion channels in the plasma membrane, as well as by mobilization of Ca²⁺ from intracellular stores. One of the best-studied examples of Ca²⁺-dependent signal transduction is the depolarization of excitable cells, such as those of neuronal, cardiac, skeletal and smooth muscle tissue, which is mediated by inward Ca²⁺ and Na⁺ currents. The Ca²⁺ current is attributed to the movement of ions through N-, L-, P- and T-type Ca²⁺ channels, which are defined both pharmacologically and by their biophysical properties, including conductance and voltage sensitivity. Here we describe several fluorescent ligands for imaging the spatial distribution and localization of Ca²⁺ channels in cells, as well as Premo Cameleon Calcium Sensor, a genetically encoded, protein-based ratiometric sensor for calcium measurements. Our complete selection of Ca²⁺ indicators is described in Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19.

Fluorescent Dihydropyridine for L-Type Ca²⁺ Channels

The L-type Ca²⁺ channel is readily blocked by the binding of dihydropyridine to the channel's pore-forming α₁-subunit. To facilitate the study of channel number and distribution in single cells, we offer fluorescent dihydropyridine derivatives. The high-affinity (–)-enantiomer of dihydropyridine is available labeled with either the green-fluorescent DM-BODIPY (D7443) or the orange-fluorescent ST-BODIPY (S7445) fluorophore. Knaus and colleagues have shown that these BODIPY dihydropyridines bind to L-type Ca²⁺ channels with high affinity and inhibit the Ca²⁺ influx in GH₃ cells. For neuronal L-type Ca²⁺ channels, the (–)-enantiomers of the DM-BODIPY dihydropyridine and ST-BODIPY derivatives each exhibit a K_i of 0.9 nM. Their affinities for skeletal muscle L-type Ca²⁺ channels are somewhat lower. Although DM-BODIPY dihydropyridine exhibits a more intense fluorescence, the particularly high degree of stereoselectivity retained by the ST-BODIPY derivatives has proven useful for the in vivo visualization of L-type Ca²⁺ channels. DM-BODIPY dihydropyridine has proven effective as a substrate for functional analysis of ABC drug transporters.

BODIPY FL Verapamil

Like dihydropyridine, phenylalkylamines also bind to the α₁-subunit of L-type Ca²⁺ channels and block Ca²⁺ transport. We offer a green-fluorescent BODIPY FL derivative (B7431) of verapamil, a phenylalkylamine known to inhibit P-glycoprotein–mediated drug efflux.

The 170,000-dalton P-glycoprotein is typically overexpressed in tumor cells that have acquired resistance to a variety of anticancer drugs (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). P-glycoprotein is thought to mediate the ATP-dependent efflux or sequestration of structurally unrelated molecules, including actinomycin D, anthracyclines, colchicine, epipodophyllotoxins and vinblastine. Verapamil appears to inhibit drug efflux by acting as a substrate of P-glycoprotein, thereby overwhelming the transporter's capacity to expel the drugs. BODIPY FL verapamil also appears to serve as a substrate for P-glycoprotein. This fluorescent verapamil derivative preferentially accumulates in the lysosomes of normal, drug-sensitive NIH 3T3 cells but is rapidly transported out of multidrug-resistant cells.

Eosin Derivatives: Inhibitors of the Calcium Pump

Eosin isothiocyanate (E18) is a potent reversible inhibitor of the erythrocyte plasma membrane calcium pump, with a half-maximal inhibitory concentration of <0.2 µM. Eosin isothiocyanate also reacts irreversibly at the ATP-binding site of this calcium pump. The succinimidyl ester of carboxyeosin diacetate (C22803), a cell membrane–permeant eosin derivative, also inhibits the erythrocyte plasma membrane Ca²⁺ pump. Fluorescein isothiocyanate (F143, BODIPY Dye Series—Section 1.4) is a weaker inhibitor of the erythrocyte plasma membrane calcium pump.

Premo Cameleon Calcium Sensor

The Premo product line combines genetically encoded ion indicators and environmental sensors with efficient BacMam delivery (BacMam Gene Delivery and Expression Technology—Note 11.1) for intracellular measurements in mammalian cells. Premo Cameleon Calcium Sensor (P36207, P36208) is a ratiometric calcium-sensitive fluorescent protein that is delivered by BacMam baculovirus-mediated transduction to a variety of mammalian cell types. This content and delivery system provides an effective and robust technique for measuring Ca²⁺ mobilization in transduced cells using microplate assays or fluorescence microscopy ().

The Premo Cameleon Calcium Sensor is based on the YC3.60 version of the fluorescent protein (FP)–based sensor (cameleon) family developed by Tsien, Miyawaki and coworkers, which is reported to have a Ca²⁺ dissociation constant of 240 nM. The sensor comprises two fluorescent proteins (enhanced cyan-fluorescent protein or ECFP and Venus variant of yellow-fluorescent protein or YFP), linked by the calmodulin-binding peptide M13 and calmodulin. Upon binding four calcium ions, calmodulin undergoes a conformational change by wrapping itself around the M13 peptide, which changes the efficiency of the fluorescence resonance energy transfer (FRET) between the CFP donor and the YFP acceptor fluorophores (Figure 16.3.1). Following this conformational change, there is an increase in YFP emission (525–560 nm) and a simultaneous decrease in CFP emission (460–500 nm) (Figure 16.3.2), making Cameleon an effective reporter of calcium mobilization. The ratiometric readout of Premo Cameleon Calcium Sensor—an increase in YFP emission (535 nm, green-yellow emission) and a decrease in CFP emission (485 nm, blue emission)—reduces assay variations due to compound or cellular autofluorescence, nonuniform cell plating, differences in expression levels between cells, instability of instrument illumination and changes in illumination pathlength.

The Premo Cameleon Calcium Sensor is designed to readily and accurately detect intracellular calcium flux from different receptors. Standard pharmacological assays for multiple GPCR agonists and antagonists have been tested. An example of the robustness and reproducibility and accuracy of the system is demonstrated using the endogenous histamine receptor in conjunction with histamine, pyrilamine, and thioperamide in HeLa cells (Figure 16.3.3). Expression levels will be maintained for several days, enabling iterative assays to be run; for instance, when examining agonist/antagonist relationships on the same cells.

Amiloride Analogs: Probes for the Na⁺ Channel and the Na⁺/H⁺ Antiporter

Amiloride is a compound known to inhibit the Na⁺/H⁺ antiporter of vertebrate cells by acting competitively at the Na⁺-binding site. The antiporter extrudes protons from cells using the inward Na⁺ gradient as a driving force, resulting in intracellular alkalinization. In 1967, Cragoe and co-workers reported the synthesis of amiloride and several amiloride analogs, which are pyrazine diuretics that inhibit the Na⁺ channel in urinary epithelia. Since then, more than 1000 different amiloride analogs have been synthesized and many of these tested for their specificity and potency in inhibiting the Na⁺ channel, Na⁺/H⁺ antiporter and Na⁺/Ca²⁺ exchanger. Unmodified amiloride inhibits the Na⁺ channel with an IC₅₀ of less than 1 µM. Additionally, amiloride is an important tool for studying the Na⁺/H⁺ antiporter. Structure–activity relationships have demonstrated that amiloride analogs with hydrophobic groups in the drug are the most potent and specific inhibitors for the Na⁺/H⁺ antiporter. For example, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, E3111) is 200-fold more potent than amiloride for inhibiting this antiporter.

Ouabain Probes for Na⁺/K⁺-ATPase

Ouabain is a member of a class of glycosylated steroids collectively known as cardiac glycosides due to their therapeutic efficacy in the treatment of congestive heart failure. Ouabain achieves this effect by binding to the catalytic α-subunit of Na⁺/K⁺-ATPase and inhibiting its transport of Na⁺ across the plasma membrane. 9-Anthroyl ouabain (A1322) is useful for localizing Na⁺/K⁺-ATPase and for studying its membrane orientation, mobility and dynamics. Anthroyl ouabain has also been employed to investigate Na⁺/K⁺-ATPase's active site, inhibition and conformational changes, as well as to investigate the kinetics of cardiac glycoside binding. BODIPY FL ouabain (B23461) has been used in combination with Alexa Fluor 555 cholera toxin B (C22843, Probes for Following Receptor Binding and Phagocytosis—Section 16.1) for visualizing Na⁺/K⁺-ATPase and ganglioside G_M1 domain localization in lymphocyte plasma membranes.

Using BacMam Technology to Deliver and Express Sodium Channel cDNA

Sodium 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, including the Nav1.2 cDNA (B10341) and the Nav1.5 cDNA (B10335).

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. Furthermore, 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, transient expression systems such as the BacMam gene delivery and expression system 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.

Glibenclamide Probes for the ATP-Dependent K⁺ Channel

Glibenclamide blocks the ATP-dependent K⁺ channel, thereby eliciting insulin secretion. We have prepared the green-fluorescent BODIPY FL glibenclamide (BODIPY FL glyburide, E34251) and red-fluorescent BODIPY TR glibenclamide (BODIPY TR glyburide, E34250) as probes for the ATP-dependent K⁺ channel. BODIPY TR glibenclamide has been used to detect sulfonylurea receptors associated with ATP-dependent K⁺ channels in bovine monocytes and in β-cells  and to label a novel mitochondrial ATP-sensitive potassium channel in brain.

The sulfonylurea receptors of ATP-dependent K+ channels are prominent on the endoplasmic reticulum (ER). Therefore, because these probes are also effective live-cell stains for ER, BODIPY FL glibenclamide and BODIPY TR glibenclamide are also referred to as ER-Tracker Green and ER-Tracker Red, respectively; see Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4 for a description of this application. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling with these probes.

FluxOR Potassium Ion Channel Assay

The FluxOR Potassium Ion Channel Assay Kits (F10016, F10017) provide a fluorescence-based assay for high-throughput screening 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 16.3.4). 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 (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. 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 high-throughput screening assay.

Each FluxOR Potassium Ion Channel Assay Kit contains:

• FluxOR reagent
• FluxOR assay buffer
• PowerLoad concentrate
• Probenecid
• 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 BacMam hERG reagent  (B10019, B10033; see below) (Figure 16.3.5).

Using BacMam Technology to Deliver and Express Potassium Channel cDNA

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 16.3.6), 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. Furthermore, 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.

Stilbene Disulfonates: Anion-Transport Inhibitors

We offer three stilbene disulfonates that have been employed to inhibit (frequently irreversibly) anion transport in a large number of mammalian cell types:

• DIDS (D337)
• H₂DIDS (D338)
• DNDS (D673)

Our stilbene disulfonate probes, which are 95–99% pure by HPLC, have significantly higher purity and more defined composition than those available from other commercial sources. DNDS was among the inhibitors used to characterize three different anion exchangers in the membranes of renal brush border cells and to compare these exchangers with the band-3 anion-transport protein of erythrocyte membranes.

These stilbene disulfonates can, in some cases, bind specifically to proteins that are not anion transporters. For example, DIDS and H₂DIDS complex specifically with the CD4 glycoprotein on T-helper lymphocytes and macrophages, blocking HIV type-1 growth at multiple stages of the virus life cycle.

DiBAC₄(5)

The membrane potential–sensing dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC₄(5), B436) initially inhibits Cl^– exchange with an IC₅₀ of 0.146 µM. However, this inhibition increases with time to an IC₅₀ of 1.05 nM, making DiBAC₄(5) a more potent inhibitor than DIDS, which has an IC₅₀ of 31 nM under similar conditions.

Eosin Maleimide

Although usually selectively reactive with thiols, eosin-5-maleimide (E118, Thiol-Reactive Probes Excited with Visible Light—Section 2.2) is known to react with a specific lysine residue of the band-3 protein in human erythrocytes, inhibiting anion exchange in these cells and providing a convenient tag for observing band-3 behavior in the membrane. Eosin-5-isothiocyanate (E18) has similar reactivity with band-3 proteins.

Premo Halide Sensor

The fluorescent protein–based Premo Halide Sensor (P10229) is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels and their modulators in cells. Chloride channels are involved in cellular processes as critical and diverse as transepithelial ion transport, electrical excitability, cell volume regulation and ion homeostasis. Given their physiological significance, it follows that defects in their activity can have severe implications, including such conditions as cystic fibrosis and neuronal degeneration. Thus, chloride channels represent important targets for drug discovery. Other methods for detecting chloride are described in Detecting Chloride, Phosphate, Nitrite, and Other Anions—Section 21.2.

Premo Halide Sensor combines a yellow-fluorescent protein (YFP) variant sensitive to halide ions with the efficient and noncytopathic BacMam delivery and expression technology (BacMam Gene Delivery and Expression Technology—Note 11.1). Premo Halide Sensor is based on the Venus variant of Aequorea Victoria green-fluorescent protein (GFP), which displays enhanced fluorescence, increased folding, and reduced maturation time when compared with YFP. Additional mutations H148Q and I152L were made within the Venus sequence to increase the sensitivity of the Venus fluorescent protein to changes in local halide concentration, in particular iodide ions. Because chloride channels are also permeable to the iodide ion (I), iodide can be used as a surrogate of chloride. Upon stimulation, a chloride channel or transporter opens and iodide flows down the concentration gradient into the cells, where it quenches the fluorescence of the expressed Premo Halide Sensor protein (Figure 16.3.7). The decrease in Premo Halide Sensor fluorescence is directly proportional to the ion flux, and therefore the chloride channel or transporter activity. Premo Halide Sensor shows a similar excitation and emission profile to YFP (Figure 16.3.8) and can be detected using standard GFP/FITC or YFP filter sets. Halide-sensitive YFP-based constructs in conjunction with iodide quenching have been used in high-throughput screening (HTS) to identify modulators of calcium-activated chloride channels.

Premo Halide Sensor (P10229) is pre-packaged and ready for immediate use. It contains all components required for cellular delivery and expression, including baculovirus carrying the genetically encoded biosensor, BacMam enhancer and stimulus buffer. Premo Halide Sensor has been demonstrated to transduce multiple cell lines including BHK, U2OS, HeLa, CHO, and primary human bronchial epithelial cells (HBEC), providing the flexibility to assay chloride permeable channels in a wide range of cellular models. To uncouple cell maintenance and preparation from cell screening, BacMam-transduced cells can be divided into aliquots and frozen for later assay. Both stable cell lines and human primary cells can be prepared frozen and “assay-ready” and can be subsequently plated as little as four hours prior to screening. Screening can be conducted in complete medium and without any wash steps. Chloride channel assays with Premo Halide Sensor are compatible with standard fluorescence HTS platforms.