Generation of Antibodies That Are Externally Acting Isoform-Specific Inhibitors of Ion Channels
There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or p
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Introduction Antibodies are proteins of the immune system that normally serve to recognize and bind foreign objects (antigens). Mutations in the antigen-binding site can produce a high number of variants with the ability to recognize a diverse range of targets. This feature can be exploited to create antibodies that display a high affinity for surface-exposed proteins such as ion channels. In addition to binding, the antibodies may modulate or down-regulate ion channel function by binding directly to the ion pore-forming subunit or to an associated regulatory subunit. An example of the success of this approach is antibody targeted to transient receptor potential polycystin 2 (TRPP2) which inhibits calcium entry evoked by shear stress (1). Another example is antibody to the N-terminus of stromal interaction molecule 1 (STIM1) which inhibits calcium entry associated with store-depletion (2). Both of these antibodies target
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_19, © Springer Science+Business Media, LLC 2013
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Jacqueline Naylor and David J. Beech
extracellular peptides. Such extracellular targeting has advantages of ease of use in living cell/tissue studies and enhanced likelihood of specificity because only extracellular epitopes are available to the antibody when cells are intact. The antibody effects are relatively rapid (within 30 min) and thus they confer an additional advantage relative to other slower methods, such as gene modification or RNA interference, which carry a greater risk of compensatory effects and the associated more complex interpretation of the arising data. Channel-blocking antibodies can be targeted to intracellular regions, but this requires complex delivery methods in living cell situations. An antibody targeted to an intracellular epitope on the C-terminus of Kir2.1 was, for example, coupled to carrier peptides to allow passage into the cell; this approach was used to demonstrate a functional role of Kir2.1 in the rat retina (3). In comparison with established ion channel modulators such as naturally occurring toxins or synthetic compounds, antibody blockers have the advantage of exceptionally high specificity and the ability to distinguish between ion channel subtypes. Such specificity was demonstrated for NESOpAb, a Na+ channel (NaV1.5) blocking antibody that selectively inhibits current carried by the neonatal splice variant of the channel, which differs from the adult form by six amino acids (4). NESOpAb may have therapeutic applications in breast cancer because the neonatal channel variant is up-regulated in cancer tissue (5). The potential of this type of antibody was also indicated by an antibody targeted to a region of the ion-conducting pore of the voltage-dependent Ca2+ channel (the a1A subunit) that is only exposed upon depolarization (6). The design of antibody channel inhibitors has been rationalized to specifically target the third extracellular loop (E3) of six membrane-spa
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