Peptide‐Specific Antibody as a Tool to Evaluate TRPM3 Ion Channel Function

Abstract

Calcium channel blockers can be used to selectively inhibit calcium influx across a membrane, making them potentially useful as therapeutic tools. Existing transient receptor potential (TRP) channel blockers are relatively non-specific, but have previously been used to characterise individual members of the TRP family. The activation of TRP channels can be more specifically inhibited through the use of anti-peptide antibodies targeted to particular regions of the channel structure (Xu & Beech, 2001. Circ. Res. 88:84–87). The polyclonal antibody TM3E3 was designed to specifically bind to a peptide corresponding to the 3rd extracellular loop of TRPM3, a member of the melastatin-like TRP family. The location and sequence of the peptide were determined using hydrophilicity analysis of the channel sequence (Xu et al., 2005. Nat. Biotechnol. 23:1289–1293). Here we use Ca2+-imaging to study the specificity of TM3E3 for human TRPM3 expressed in HEK-293 cells. Extracellular application of the antibody has a functional effect, inhibiting the sphingosine-induced activation of TRPM3. It does not inhibit activation of the closely related channel TRPM2 or the canonical family member TRPC5. TM3E3 will serve as a useful tool for further characterisation of endogenous TRPM3 in a variety of cells and tissues, and more importantly the results strengthen the idea that E3 targeting may be used to generate specific ion channel blockers for a variety of channels, with therapeutic potential. This work is supported by the BBSRC, the Welcome Trust and the BHF. Many thanks to C. Harteneck for providing the TRPM3 clone.