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Zhu MX, editor. TRP Channels. Boca Raton (FL): CRC Press/Taylor & Francis; 2011.

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Chapter 7Assessing TRPC Channel Function Using Pore-Blocking Antibodies



There are ~23,000 human protein-coding genes. One third of them encode membrane proteins including ion channels, receptors, transporters, and exchangers.1,2 To understand the physiological function of individual genes, the development of specific tools targeting the gene of interest is essential. The technology by gene modification, such as antisense oligonucleotides, small interfering RNA (siRNA), gene knockout, or transgenic animals, has provided useful approaches to reveal individual gene function; however, some limitations of these techniques are inevitable. Among them, the knockdown of protein expression takes time (at least days) and will likely cause compensatory changes that obscure the interpretation of the research findings. The study of cell surface receptors and ion channels has benefited greatly from the use of small molecule blockers and activators, which cause acute modulation of the protein function, allowing direct comparison of the sample before and after the drug treatment. This often provides a clear answer as to whether the target protein is involved in the physiological process being studied without a concern of the compensatory effect.

The traditional approach for identifying a specific tool for ion channel study is mainly based on screening synthetic chemicals or natural compounds, which, during the past few decades, has provided some reliable research tools and effective therapeutic drugs, such as tetrodotoxin that blocks voltage-gated Na+ channels, dihydropyridines and cone snail toxins for voltage-gated Ca2+ channels, and many receptor agonists or antagonists. Screening chemical compounds is undoubtedly useful; however, the process is laborious and time-consuming, and there is no guarantee that a specific and potent drug will be found. Therefore, more targeted approaches would appear to be necessary in order to match the fast pace of new ion channel discovery.

Antibodies are renowned for their exquisite specificity and unlimited diversity.3 Therefore, we have tried to develop a new class of antibodies targeting ion channel pore regions, for example, the third extracellular loop of TRPC channels, called E3-targeting antibodies, in order to functionally interfere with channel properties.4, 5 Not only can these E3-targeting antibodies be used as ordinary antibodies for protein labeling, Western blotting, immunostaining, and immunoprecipitation, but they are also useful as specific pharmacological tools for in vitro or even in vivo functional studies. Since our reports on the methodology and applications,48 several groups have tried the method, and some pore-blocking antibodies have been successfully developed or reproduced, such as the pore-blocking antibodies for TRPC channels,9,10 Eag1 potassium channels,11 TRPV1 channels,12 TRPM3 channels,13 and CaV1.2 channels.14

Given the huge potential use of pore-blocking antibodies in ion channel research, this chapter describes the generation of functional antibodies and their applications including how to design a functional antibody, how to screen the antibodies, how the antibody can be used as a pharmacological tool (especially for assessing the TRPC function), and what the advantages and limitations are.


Antibodies that can alter the ion channel function via direct binding or interaction with the ion channel protein are functional antibodies. Besides the pore-blocking antibodies we reported,4 functional modulation by antibodies targeting the cytoplasmic protein regions has also been described, such as the C-terminal antibodies for a potassium channel (KV1.2)15 and the inositol 1,4,5-trisphosphate receptor (IP3R),16 as well as the N-terminal antibody for the stromal interaction molecule 1 (STIM1).17 Antibodies targeting the alpha subunit of G-proteins18 or T-tube membrane19 also indirectly change the ion channel function.

The development of pore-blocking antibodies could be a simple and straightforward approach for stopping ion flow via a specific ion channel and useful for studying individual channel function in native cells, especially for these ion channels lacking specific blockers.20 The E3-targeting methodology is for ion channels that contain six transmembrane segments and three extracellular loops for each subunit, such as TRPCs and shaker potassium channels.4 The strategy can be easily adopted for other ion channel families with three extracellular loops, such as TRPVs, TRPMs, TRPA1, and cyclic nucleotide-gated (CNG) ion channels,20 or even for other ion channels with just one or two extracellular loops, such as inwardly rectifying potassium channels and ORAI channels.17,21 In order to help researchers achieve a high success rate for pore-blocking antibody generation, some principles are given as follows.

7.2.1. Ion Channel Topology Analysis

The structure of TRPCs is similar to the well-described Shaker potassium channels including six-membrane spanning segments (S1–S6), a putative channel pore region located between S5 and S6, and the intracellularly located N- and C-termini. The hydropathy plot and the structure prediction software, e.g., the Expert Protein Analysis System (ExPASy) on the server of the Swiss Institute of Bioinformatics, are helpful for understanding a new ion channel topology.

7.2.2. Selection of Target Epitope

The selection of antigenic peptide is a critical step for successful generation of a pore-blocking antibody. Sequence alignment with other related isoforms is essential for the selection of isoform-specific epitopes, such as those used for TRPC15 and TRPC5.4 These E3 epitopes are very close to or even partially overlap with the putative ion selectivity filter (Figure 7.1). There is software, such as Lasergene, to help predict peptide antigenicity. The following principles should be considered.

FIGURE 7.1. E3-targeting methodology.


E3-targeting methodology. (a) Hydropathy plots of ion channel subunits with six transmembrane segments (S1–S6). Shaker is a Drosophila K+ channel, and KV1.2 and KV3.1 are mammalian homologs. TRPC1 and TRPC5 show a similarity structure to the K (more...) Antibody Accessibility

The selected target region for antibody generation should be accessible by IgG and is potentially important for the ion channel function. For example, the third extracellular loop near the channel pore region is important for the TRPC channel function. The cysteine8 and negatively charged glutamate (Glu)22 residues are functionally important for TRPC5 channel activity. Some ion channels may be glycosylated, and therefore, the potential glycosylated site should be avoided if possible because antibodies targeted to the peptide sequences may not recognize the modified native protein. Hydrophilicity and Flexibility

The hydrophilic regions tend to reside on the surface of membrane proteins, while the hydrophobic regions tend to be found hidden in the interior of the protein structure or in the membrane. Therefore, the hydropathy plot is a helpful guide for epitope selection. If a hydrophilic region is selected, then peptide solubility should not be a problem. If there is a choice, one should select an antigenic peptide with as few hydrophobic residues (e.g., trytophan, valine, leucine, isoleucine, and phenylalanine) as possible. Glutamines may also cause insolubility, as it can form hydrogen bonds between peptide chains, so multiple glutamines in an epitope should be avoided. The introduction of proline or tyrosine can induce structural motifs, thereby enhancing the immunogenic potential of the peptide. In addition, antibodies tend to bind with higher affinity to those epitopes that are flexible enough to move into accessible positions. However, unlike the C- or N-terminal ends of the channel, the transmembrane regions may have less flexibility. Length of Epitope

The length of the epitope is also important. Longer antigenic peptides may have a greater conformational similarity to the native protein and are therefore more likely to induce antibodies that recognize the natural protein. However, if an epitope is too long, it may lose its specificity, especially when targeting the extracellular pore region. Data suggest that a single antigenic determinant, i.e., the smallest immunogenic peptide, is between five and eight amino acids, and therefore, a length of 15–20 amino acids is preferable for designing an antigenic peptide, as it should contain at least one epitope and adopt a limited number of conformations. Like other peptide-based antibody generations, a cysteine should be added to the end of the selected epitope for antibody affinity purification purpose if there is no internal cysteine in it.

7.2.3. Antigenic Peptide Synthesis and Animal Immunization

The selected epitope should be confirmed to be unique by BLAST searching against the GenBank protein database before sending the sequence for peptide synthesis. The integrity and purity of the peptide should be assayed with high-performance liquid chromatography. If the purity is less than 80%, it should not be used because low-purity peptides will likely yield poor-quality antibodies for pore-blocking functional studies.

Animal immunization is laborious work. However, many companies provide an antibody production service, such as the standard 77-day protocol for rabbit polyclonal antibody generation (Table 7.1). Briefly, rabbits approximately 2–3 kg are usually used for immunization. The synthetic peptide should be completely dissolved in a sterile saline at a concentration of 100–500 g/mL. About 1 mL of antigen solution is made into an emulsion with an equal amount of complete Freund’s adjuvant (CFA) (1 mg of dried Mycobacterium tuberculosis H37Ra, 0.85 mL of paraffin oil, and 0.15 mL of mannide monooleate). Then 1 mL of the emulsion is injected into the popliteal lymph nodes, and the remaining emulsion (~1 mL) can be subcutaneously injected into numerous small depots arranged along the spine.



Schedule for Immunization and Bleedinga

The booster and bleeding protocol is also listed in Table 7.1. The incomplete Freund’s adjuvant (IFA) (0.85 mL of paraffin oil and 0.15 mL of mannide monooleate) is used after the first CFA injection. The test bleeds will start at the fifth week of the protocol and terminate at day 77 or a later day depending on the antibody titration tested by an enzyme-linked immunosorbent assay.

7.2.4. Pore-Blocking Antibody Screening Enzyme-Linked Immunosorbent Assay

The immune serum should be screened by ELISA. The successful antibodies usually have a reasonable or high titration, for example, the anti-TRPC1 antibody (T1E3) had a titration higher than 1:50,000 dilution. The detailed procedure for ELISA is given as follows.

Coating antigen: Antigenic peptide dissolved in 50 mM Na2HCO3 (pH 9.6) at the concentration of 4 g/mL is used for plate coating. For example, for the 96-well Nunc-Immuno Plate, 50 L of the peptide solution is added to each well, and the plate is covered with a piece of paraffin membrane and kept at 4°C overnight.

Blocking nonspecific binding: The coated plate is washed three times with phosphate buffered saline (PBS) containing 0.2% Tween20 (PBS/Tween20) followed by an incubation with 300 L per well 1% dry milk in PBS at 37°C for 1 hour to block the nonspecific binding sites.

Incubation with antibody: The plate is then washed three times with PBS/ Tween20 before the addition of 50 L of serial diluted antiserum (1:50 to 1:50,000 dilutions) to the wells. The plate is then incubated at 37°C for 2 hours and washed three times with PBS/Tween20 again. The secondary antibody (goat anti-rabbit IgG conjugated with horseradish peroxidase at 1:5,000 dilution) is added at 50 L/well and the plate incubated at 37°C for 1 hour.

Color development: After washing the plate with PBS/Tween20 three times, 50 L of the color development solution ((2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt 5.5 mg dissolved in a 10-mL phosphate citrate buffer plus 1 μL H2O2) is added into each well. The plate is gently shaken on a rotator and incubated at room temperature for 30 min in dark for color development, followed by absorbance reading at a wavelength of 405 nm using a plate reader. Western Blotting and Immunostaining

Western blotting is useful for examining the specificity of antibody binding to the target protein. Cell lysates prepared from both native tissues and cultured cells transfected with the corresponding cDNA for the target protein should be used for validation of the pore-blocking antibody. In addition, binding to the extracellular region can be confirmed by immunocytochemistry performed on both permeabilized and nonpermeabilized cells. The extracellular binding antibody is expected to give positive staining regardless of whether the cells are permeabilized. Fluorescence-Activated cell sorting (FACS)

Fluorescence-activated cell sorting is a powerful tool for screening the pore-blocking antibodies. The extracellular binding of E3-targeting antibodies can be examined by FACS using transfected cells or native cells if the channel is well expressed. For example, E3-targeting TRPC antibodies can be tested using the TRPC-transfected HEK-293 cells through a secondary antibody conjugated with fluorescent probes.

7.2.5. Antibody Purification

Pore-blocking antibody should be purified. The protein A column and the immobile peptide column are used for purification. This step is essential, especially for these lower titration antisera. The solvent in the purified antibodies should be completely removed by dialysis in PBS. The purified antibodies are stored in a –80°C freezer in aliquots. Affinity Purification of IgG Using Protein A

The protein A column can be used for purification of rabbit polyclonal antibodies. For example, the HiTrap affinity column (Amersham Pharmacia Biotech) has a high binding capacity for IgG. Briefly, the column is prewashed with 10 column volumes of the binding buffer (20 mM sodium phosphate, pH 7.4) and then loaded with rabbit serum samples diluted at 1:1 dilution in the binding buffer. The column is washed with 10 column volumes of the binding buffer or until no material appears in the effluent or the absorbance of the effluent at 280 nm approaches the background level. The bound IgG is then eluted from the column with four column volumes of the elution buffer (0.1 M citric acid). The eluted IgG is collected in tubes with the neutralization buffer (1 M Tris-HCl, pH 9.0). The eluted immunoglobulin is measured by a spectrophotometer at the wavelength of 280 nm. For mouse IgG purification, the protein G column should be used instead. Affinity Purification Using Immobile Peptide Column

The immobile peptide column is prepared by conjugating the resin (SulfoLink coupling gel, Pierce Chemical Company, Rockford, Illinois, USA) with the antigenic peptide. The resin is prewashed with a coupling buffer (50 mM Tris and 5 mM EDTA, pH 8.5) and then mixed with an equal volume of 0.1% antigenic peptide in the coupling buffer. The mixture is rotated on a platform for 1 hour at room temperature and washed three times with the coupling buffer, each followed by centrifugation. The resin is then saturated by incubation with 50 mM cysteine, which is followed with washing in a wash buffer (1 M NaCl in PBS).

For antibody purification, an equal volume of PBS is mixed with the antiserum or the crude antibody and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant is then mixed with peptide-conjugated resin and incubated at 4°C overnight. The resin is loaded onto a small column, washed with the wash buffer, and then eluted using 200 mM glycine-HCl. The flow-throughs before and during the elution are collected into a series of test tubes containing the neutralization buffer. Aliquots of the eluted samples are measured at 280 nm and further tested by ELISA for the presence of proteins and desired antibodies, respectively.

7.2.6. Functional Test of Pore-Blocking Antibodies Patch-Clamp Recording

The blocking effect of the antibody should be tested by patch clamp recordings using either native or transfected cells that express the channel of interest, following examples for the TRPC pore-blocking antibodies4,17 and the potassium channel antibodies.23 Proper controls should be set in parallel, such as the antibody pre-incubated with the antigenic peptide to block the binding site, pre-immune IgG, and peptide alone. The dose-response curve may be useful for determining the antibody concentration needed for functional study. New patch-clamp recording systems may also be useful, for example, the planar patch;17 however, the detached cells should be maintained at a good condition for high-quality recordings. Calcium Imaging

For calcium-permeable channels, the pore-blocking effect can be tested using Ca2+ imaging, as exemplified for the TRPC1 and TRPC5 blocking antibodies.4–6 Again, proper controls should be carried out in parallel or in alternating orders with the test samples in order to minimize the influence of experiment-to-experiment variations of Ca2+ imaging assay.

The FlexStationTM calcium assay system provides a fast, simple, and reliable fluorescence-based assay for detecting changes of intracellular Ca2+ concentrations. (See Chapter 1 for more details.) This high-throughput system should be effective for screening pore-blocking antibodies and/or new chemical ligands of ion channels.


7.3.1. Assessing Ca2+ Influx TRPC1 Forms Store-Operated Channel Subunit

The pore-blocking antibody for TRPC1 (T1E3) was first applied to the freshly isolated rabbit cerebral arterioles to investigate its role in store-operated Ca2+ influx in the smooth muscle cells.5 Using a Ca2+-refilling protocol following store depletion by pretreatment of cells with thapsigargin, the store-operated Ca2+ influx was shown to be significantly inhibited by incubation with T1E3 (Figure 7.2), an effect that was prevented by pre-absorption of the antibody using the antigenic peptide absorbed. This experiment gave the first direct evidence that TRPC1 confers store-operated Ca2+ influx in vascular smooth muscle.5 We suggested that TRPC1 could be a subunit of the store-operated channels or it could constitute a subpopulation of these channels because the inhibition of the store-operated Ca2+ influx by T1E3 was only around 25%. The larger part of the remaining T1E3-insenstive store-operated Ca2+ entry in the smooth muscle cells could be mediated by other channels. As we have postulated, several store-operated Ca2+ entry pathways have recently been identified in vascular smooth muscle cells. These include heteromultimeric TRPC1/5 channels with store-operated properties6,24 and Ca2+ entry mediated through STIM117,25 and Orai1.26 The contribution of TRPC1 to the store-operated Ca2+ influx has also been demonstrated in TRPC1 knockout mice27 and in cells using the siRNA approach to knock down TRPC1 expression.2830

FIGURE 7.2. TRPC1 pore-blocking antibody T1E3 inhibits store-operated Ca2+ entry in freshly isolated arterial smooth muscle cells.


TRPC1 pore-blocking antibody T1E3 inhibits store-operated Ca2+ entry in freshly isolated arterial smooth muscle cells. (a) Ca2+ influx was measured by switching the external solution from 0 to 1.5 mM Ca2+ to arterial cells treated with and without thapsigargin. (more...)

We have not tested the T1E3 antibody in TRPC1-transfected cells because the TRPC1 current in the overexpression system is relatively small and very difficult to distinguish from the endogenous activity, despite the fact that the TRPC1 protein expression and subcellular localization have been confirmed in HEK293 cells and tsA 201 cells by the FLAG- and EYFP-tagged proteins, respectively, and by T1E3 antibody labeling (S. Z. Xu, unpublished data). It remains a mystery why the over-expressed TRPC1 cannot produce a remarkable current. The potential explanation could be due to its relatively high endogenous expression in many cell types and/or its lack of ability to traffic to the plasma membrane and assemble into store-operated channels when expressed alone. Assessment of TRPC5 Function

The overexpressed TRPC5 channel has a typical outward–inward–outward rectification (“N” shaped) current–voltage (I–V) relationship31 and can be activated by multiple mechanisms including a G-protein coupled receptor pathway and internal Ca2+ store depletion.32 The TRPC5 channel can also be directly activated by lanthanides (Gd3+ and La3+)22 and some reducing agents (thioredoxin, Tris[2-carboxyethyl] phosphine (TCEP), and dithiothreitol (DTT))8 via modification of glutamate residues and a disulfide bridge in the S5–S6 region, respectively. The human TRPC5 current can be abolished by 2-APB.31 Application of TRPC5 pore-blocking antibody (T5E3) also significantly inhibited the TRPC5 current (Figure 7.3). The effect is specific for TRPC5 because the pore-blocking antibody T5E3 has no effect on cells expressed with TRPC6, and the antigenic peptide-absorbed antibody did not show any effect.6 The store-operated Ca2+ entry has been assessed in the rabbit arteriole using T5E3. The store-operated Ca2+ entry is significantly inhibited by T5E3, but no effect was observed in the arterioles without store depletion, suggesting that TRPC5 contributes to store-operated Ca2+ entry in the native cells.6

FIGURE 7.3. Pore-blocking antibody T5E3 inhibits the TRPC5 current.


Pore-blocking antibody T5E3 inhibits the TRPC5 current. (a) Whole-cell current evoked by 10 μM gadolinium (Gd3+) in HEK293 cells with inducible expression of human TRPC5 by tetracycline (HEK-TRPC5). Current–voltage relationship for TRPC5 (more...)

The blocking effect of T5E3 has also been examined in cerebral arterioles treated with La3+ because lanthanides have a unique stimulating effect on TRPC5 channels. After perfusion with 50 μM La3+, the response to La3+ in the store-depleted cerebral arteriole displayed two phases, i.e., an initial inhibitory phase and a secondary stimulatory phase as the intracellular Ca2+ concentration gradually rose above the baseline.4 T5E3 enhanced the inhibition of La3+ on the initial phase, which is presumed to be a store-operated component, and inhibited the stimulating phase of La3+, which could be mediated by the homomeric TRPC5 channel.4 These data suggest that native TRPC5 may also constitute a store-operated channel or contribute to the store-operated Ca2+ influx in smooth muscle cells. Store-Operated TRPC1/5 Heteromultermeric Channel

The endogenous store-operated channel in cerebral arteriolar smooth muscle cells shows a tendency toward outward rectification,6 which is quite different from the inward rectification or the “N”-shaped I–V curve seen for the overexpressed human TRPC531,32 and mouse TRPC5 channels.3338 Therefore, it is unlikely that the native store-operated channel is formed by homomeric TRPC1 or TRPC5 channels. Because overexpressed TRPC5 forms complexes with TRPC1 and heterologous co-expression of TRPC1 and TRPC5 gives rise to currents with outward rectification,24,34 we tested the idea that the native store-operated channel in vascular smooth muscle could be a complex of TRPC5 and TRPC1. We found that TRPC1 immunoprecipitated with TRPC5 from cell lysates prepared from human saphenous vein and from tsA 201 cells co-expressing TRPC5 and TRPC1. The I–V curve of store-operated currents in the TRPC1/TRPC5 co-expressing cells is similar to that recorded from arteriolar smooth muscle cells. Therefore, we suggested that TRPC1/5 heteromultimeric channels could be an important component for the SOC in native smooth muscle cells. Other Store-Operated Channels

STIM1 and ORAI1 have been reported to contribute to store-operated Ca2+ entry in vascular smooth muscle cells.26,39 Recently, functional interactions among STIM, ORAI, and TRPC proteins have been demonstrated in several studies,4045 although independent signaling and localization of TRPCs and STIM1/ORAI1 have also been described.46 An extracellular anti-STIM1 antibody significantly inhibited the store-operated current in the smooth muscle cells,17 suggesting that plasma membrane STIM1 protein contributes to store-operated Ca2+ entry. This further demonstrates the utility of functional antibodies as tools for exploring ion channel functions in native cells.

7.3.2. Assessing Native Cell Function Smooth Muscle Cell Proliferation and Migration

Proliferation and migration are two important non-contractile properties of smooth muscle cells. They are involved in neo-intimal growth, which is a key pathological process for coronary artery re-stenosis that usually happens after cardiac angioplasty surgery. The pore-blocking antibody T1E3 has been used as a specific tool for studying smooth muscle cell proliferation. Incubation with T1E3 significantly inhibited neo-intimal growth in human saphenous vein, and the effect was confirmed by transfection of TRPC1 antisense oligos.47 In addition, proliferation of the smooth muscle cell line A7r5 was also inhibited by T1E3, while the control pre-immune serum had no effect on cell proliferation.47

To study cell migration, the wound injury model is used for the primary cultured smooth muscle cells.24 Sphingsine-1 phosphate (S1P) can significantly induce smooth muscle migration. This effect is mediated by the TRPC5 channel because the incubation with the TRPC5 blocking antibody (T5E3) inhibited the S1P-induced migration, an effect also confirmed by transfection with a dominant negative TRPC5 mutant (DN-TRPC5).24 TRPCs Regulate Cell Secretory Function

Ca2+ is important in the regulation of cell secretion. The involvement of TRPCs in the cell secretory function has been described in salivary gland epithelial cells for fluid secretion,27,48 GnRH neurons for gonadotropin-releasing hormone (GnRH) secretion,49 and rat pituitary cells for adrenocorticotropin release.50 The basal secretion of alkaline phosphatase is enhanced in COS-7 cells transfected with the cDNA for TRPC3 or TRPC7, but not that for TRPC1.51

Using TRPC1 and TRPC5 pore-blocking antibodies, it was shown that the levels of metalloproteinases MMP2 and MMP9 secreted by fibroblast-like synoviocytes (FLS cells) were significantly increased by the treatment of T1E3 and T5E3, suggesting that TRPC1 and TRPC5 channels are involved in the cell secretory function. This increasing effect by TRPC channel blockade has been confirmed by transfection with TRPC1 and TRPC5 siRNAs, suggesting that the constitutive and endogenous threodoxin-induced TRPC5 channel activity is essential for regulating cell secretion.8 TRPC Channel and Smooth Muscle Contraction

The role of TRPCs in smooth muscle contraction is still unclear, especially owing to the lack of in vivo experimental data. Application of the TRPC1 pore-blocking antibody for investigating smooth muscle contractility was reported in 2003.7,52 Interestingly, the endothelin-1 induced contraction in rat tail artery was significantly inhibited by T1E3, but no effect was observed in the basilar arteries.7 The reason is unclear, probably owing to the interaction with lipid raft or the differential expression of TRPCs in these tissues.7

TRPC6 is an important and highly expressed TRP isoform in vascular smooth muscle cells.53,54 TRPC6 antisense oligodeoxynucleotides inhibited contraction in the organ cultured cerebral arteries.55 On the contrary, elevated blood pressure was reported in TRPC6 knockout mice, which has been explained as due to compensation by TRPC3.56

The upregulation of the TRPC gene or protein expression has been reported in hypertension, such as TRPC6 in hypertensive rats,57 TRPC3 and TRPC6 in idiopathic pulmonary hypertensive patients,58,59 and TRPC3 and TRPC5 in essential hypertension.60 These data suggest that TRPCs are important for regulating vascular tone, providing a clue that TRPCs could be good new molecular targets for antihypertensive therapy.


There are several advantages for application of pore-blocking antibodies: (1) The successful antibody can be used as a pharmacological tool for acute experiments in vivo. (2) Unlike synthetic chemicals, the pore-blocking antibodies are usually nontoxic and could be introduced for in vivo experiments. (3) The procedures for antibody generation are well established, so it should be a simple and quick way to make an antibody tool. (4) The pore-blocking antibody also can be used as an ordinary antibody for Western blotting, immunolabeling, immunoprecipitation, and ELISA if successful.

The disadvantages include the following: (1) Antibodies are unstable, and therefore, the proper storage and transportation are important. (2) Large-scale production of the antibody could be a problem, resulting in the limited use. (3) Batch-to-batch variations exist, calling for antibody titration for each batch. (4) Generation of a monoclonal antibody is still costly, and the affinities of monoclonal antibodies are generally lower than polyclonal antibodies. (5) Antibodies sometimes display unexpected cross reactions with unrelated antigens. (6) The ability for tissue penetration by the antibody should be considered if used for in vivo studies.


Many endogenous autoantibodies targeting ion channels have been identified; some of them are related to disease development. For example, antibodies to voltage-gated calcium, potassium channels, and glutamate receptors have been detected in the sera and cerebrospinal fluids of patients with ataxia, limbic encephalitis, and certain forms of epilepsy.6163 Some autoantibodies could be stimulatory, such as GluR3 autoantibodies.64 Generally, the autoantibodies are regarded as pathogenic factors. Recently, a TRPC3 autoantibody has been reported in patients with myasthenia gravis, and this could contribute to the contractile abnormalities of the skeletal muscle.65

Therapeutic antibodies targeting endogenous proteins have been reported to treat a range of noninfectious diseases, such as cancer,66 Alzheimer’s disease,67 and stroke.68 Some targets are membrane proteins, such as the VEGF receptor,69 suggesting that antibodies targeting specific endogenous protein or protein segment are of potential therapeutic significance. Although the pore-blocking antibodies for TRPC channels have not been tested in in vivo studies, there is a great potential for them in therapeutic development if TRPCs are linked to human diseases.


The pore-blocking antibodies can be used as powerful pharmacological tools to explore the ion channel function, especially for studying the native cell function via acute application. Unlike the procedures using siRNA or gene knockout, the blocking antibody is thought to directly interact with the channel pore and stop the ion flow. The E3-targeting methodology provides a foundation for pore-blocking antibody generation targeting ion channel subunits with six transmembrane segments. However, this strategy can be adopted for other ion channels, for instance, in the case of channels with one or two extracellular loops or even no loop, but an extracellular N-terminus. Although there is no report of a therapeutic antibody specifically developed for targeting ion channels, the successful generation and application of the pore-blocking antibodies may pave the way for further development in this field of potential immunotherapeutics for ion-channel-related diseases.


This work was supported by the British Heart Foundation and HYMS prime pump award.


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