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. 2008 Oct;155(4):567-73.
doi: 10.1038/bjp.2008.283. Epub 2008 Jul 7.

Production of a specific extracellular inhibitor of TRPM3 channels

J Naylor  1 , C J Milligan, F Zeng, C Jones, D J Beech
Affiliations

Production of a specific extracellular inhibitor of TRPM3 channels

J Naylor et al. Br J Pharmacol. 2008 Oct.

Abstract

Background and purpose: Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.

Experimental approach: A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca(2+) measurement.

Key results: Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5-10 min. Ca(2+) entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response.

Conclusions and implications: The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.

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Figures

Figure 1
Figure 1
Overexpression of melastatin transient receptor potential 3 (TRPM3) and binding of antibody. Fluorescence images of human embryonic kidney (HEK) 293 cells, with (Tet+) or without (Tet−) induction of TRPM3 expression. The upper panel shows green fluorescence (from FITC conjugated to the secondary antibody) and the lower panel blue fluorescence (from DAPI, which labels cell nuclei). Inclusion of anti-TRPM3 antibody (TM3E3 Ab) in the staining protocol is specified, where ‘+pep.' indicates pre-adsorption of TM3E3 to its antigenic peptide (10 μM). Scale bar=20 μm. Images are representative of three independent experiments.
Figure 2
Figure 2
Inhibition of ionic current. Currents were measured under voltage-clamp using whole-cell planar patch-clamp recording. The holding potential was 0 mV and 0.2-s ramp changes in voltage from −100 to +100 mV were applied every 10 s. All cells were HEK 293 cells induced to express TRPM3. (a and c) Mean currents sampled at +80 and −80 mV, each normalized to the amplitude immediately before bath-application of antiserum (TM3E3) without (a, n=8) or with (c, n=4) pre-adsorption to 10 μM antigenic peptide. Pregnenolone sulphate (PregS) was bath applied at 25 μM. TM3E3 was used at 1:500 dilution. (b and d) Typical current-voltage relationships (I–Vs) from the experiments underlying (a) and (c).
Figure 3
Figure 3
Inhibition of Ca2+ entry. Intracellular Ca2+ measurements from HEK 293 cells with (Tet+) and without (Tet−) induced expression of TRPM3. Responses to 25 μM pregnenolone sulphate (PregS) are shown. (a) Typical FlexStation experiment showing comparison of the effects of pre-incubation with dialysed TM3E3 or its pre-immune (pre.) serum (1:500 dilution). Typical of n/N=3/18. (b) Mean data showing block of the PregS response by a range of dilutions of dialysed TM3E3 antiserum, as a percentage of responses in dialysed pre-immune serum (n/N=3/18).
Figure 4
Figure 4
Inhibition of TRPM3 activated by a different agonist. Microscope-based intracellular Ca2+ measurements showing responses to 20 μM sphingosine (SPH). Comparisons are shown of the effects of pre-incubation with TM3E3 or its pre-immune (pre.) serum (1:4000 dilution) in HEK 293 cells transiently expressing (a) TRPM3-yellow fluorescent protein (YFP) (pre n/N=3/29, TM3E3 n/N=3/22), or (b) YFP alone (pre n/N=3/29, TM3E3 n/N=3/39).
Figure 5
Figure 5
Functional specificity within the TRP family. Intracellular Ca2+ measurements from HEK 293 cells with (Tet+) and without (Tet−) induced expression of (a) TRPM2 or (b) canonical transient receptor potential 5 (TRPC5). (c) Chinese hamster ovary (CHO) cells without (wt, wild type) or with stable expression of vanilloid transient receptor potential 4 (TRPV4). The different TRP channels were activated by hydrogen peroxide (a, 1 mM H2O2), gadolinium (b, 0.1 mM Gd3+) and 4α-phorbol-12,13-didecanoate (c, 10 μM 4αPDD). Cells were pre-incubated with TM3E3 or its pre-immune (pre.) serum (1:4000 dilution): typical of n=3 (N=27–34) in each case.
Figure 6
Figure 6
Lack of effect of TM3E3 on agonist-evoked Ca2+ release. Intracellular Ca2+ measurements from non-induced (Tet−) HEK 293 cells showing an endogenous response to extracellular application of 10 μM ATP. Cells were pre-incubated with TM3E3 or its pre-immune (pre.) serum (1:500 dilution): typical of n/N=3/18.
Figure 7
Figure 7
Summary of E3-targeted antibody blockers. The illustration in the lower half of the figure is the general topology of a six membrane-spanning ion channel subunit. NaV1.5 and CaV2.1/2 are concatamers of four such subunits. Names listed across the top are ion channels to which E3-targeted blocking antibodies have been successfully generated. There are double lines to TRPC1 and NaV1.5 because two different E3-targeted antibodies have been generated, each to different peptides within E3. References are: Zhou et al., 1998; Xu and Beech, 2001; Rosado et al., 2002; Chioni et al., 2005; Xu et al., 2005b; Klionsky et al., 2006; Gomez-Varela et al., 2007; Liao et al., 2008.
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References

    1. Alexander SPH, Mathie A, Peters JA.Guide to Receptors & Channels (GRAC) Br J Pharmacol 2008153Suppl 2S1–209.3rd edn. - PMC - PubMed
    1. Beech DJ, Sukumar P. Channel regulation by extracellular redox protein. Channels. 2008;1:400–403. - PMC - PubMed
    1. Brackenbury WJ, Chioni AM, Diss JK, Djamgoz MB. The neonatal splice variant of NaV1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells. Breast Cancer Res Treat. 2007;101:149–160. - PMC - PubMed
    1. Chioni AM, Fraser SP, Pani F, Foran P, Wilkin GP, Diss JK, et al. A novel polyclonal antibody specific for the NaV1.5 voltage-gated Na+ channel ‘neonatal' splice form. J Neurosci Methods. 2005;147:88–98. - PubMed
    1. Gomez-Varela D, Zwick-Wallasch E, Knotgen H, Sanchez A, Hettmann T, Ossipov D, et al. Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity. Cancer Res. 2007;67:7343–7349. - PubMed

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