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. 2018 May;365(2):227-236.
doi: 10.1124/jpet.117.245118. Epub 2018 Feb 16.

Targeting KCa1.1 Channels with a Scorpion Venom Peptide for the Therapy of Rat Models of Rheumatoid Arthritis

Mark R Tanner  1 , Michael W Pennington  1 , Brayden H Chamberlain  1 , Redwan Huq  1 , Elizabeth J Gehrmann  1 , Teresina Laragione  1 , Pércio S Gulko  1 , Christine Beeton  2
Affiliations

Targeting KCa1.1 Channels with a Scorpion Venom Peptide for the Therapy of Rat Models of Rheumatoid Arthritis

Mark R Tanner et al. J Pharmacol Exp Ther. 2018 May.

Abstract

Fibroblast-like synoviocytes (FLSs) are a key cell type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLSs and found that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects. In this study, we aimed to determine whether the KCa1.1 β1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch-clamp and functional assays were used to determine whether IbTX can regulate FLSs through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined whether IbTX causes side effects including incontinence or tremors in rats, compared with those treated with the small-molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 were expressed by FLSs in joints of rats with experimental arthritis. IbTX inhibited KCa1.1 channels expressed by FLSs from patients with RA and by FLSs from rat models of RA and reduced FLS invasiveness. IbTX significantly reduced disease severity in two rat models of RA. Unlike paxilline, IbTX did not induce tremors or incontinence in rats. Overall, IbTX inhibited KCa1.1 channels on FLSs and treated rat models of RA without inducing side effects associated with nonspecific KCa1.1 blockade and could become the basis for the development of a new treatment of RA.

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Figures

Fig. 1.
Fig. 1.
KCa1.1 is expressed on CIA-FLSs in vivo. (A–C) Example images of synovial tissue from rats with CIA that had signs of disease for 14 days stained for expression of KCa1.1α (red) and KCa1.1β3 (brown) (A), the FLS marker podoplanin (red) and KCa1.1α (brown) (B), or the FLS marker cadherin-11 (red) and KCa1.1α (brown) (C). Dashed boxes on the left demarcate the areas magnified to 80× shown on the right. Arrowheads point to double-stained cells. Scale bar, 50 μm. Original magnification, 40× (left) or 80× (right).
Fig. 2.
Fig. 2.
IbTX inhibits KCa1.1 channels expressed on FLSs and reduces ex vivo FLS invasion. (A) Representative whole-cell potassium currents (top) in RA-FLSs, PIA-FLSs, and CIA-FLSs before (gray) and after administration of 3 nM IbTX (black) and dose-response inhibition of potassium currents by IbTX (bottom). (B) Invasiveness of FLSs from patients with RA and rats with either PIA or CIA through Matrigel-coated Transwell inserts in the presence of 10 μM paxilline (Pax) or 10 μM IbTX. Data are the mean ± S.E.M. (n = 3 donors per group). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 3.
Fig. 3.
IbTX reduces signs of disease in PIA. (A) Clinical scores of paw inflammation from rats with PIA treated with vehicle (white) or 0.05 mg/kg (red), 0.5 mg/kg (gray), or 5 mg/kg IbTX (blue) subcutaneously every other day starting at disease onset. Data are the mean ± S.E.M. (n = 6 rats in the 0.05 mg/kg IbTX and 5 mg/kg IbTX groups, n = 9 rats in the 0.5 mg/kg IbTX group, and n = 15 rats in the vehicle-treatment group). (B) Clinical scores of paw inflammation from rats with PIA treated with vehicle (white), 2 mg/kg paxilline (black), or 20 mg/kg paxilline (green) intraperitoneally every other day starting at disease onset (n = 7 to 8 rats per group). (C) Hematoxylin/eosin staining (top) and safranin O/fast green staining (bottom) of tissue sections of paws from rats with PIA treated with vehicle (left) or IbTX (right). Arrowheads indicate areas of hyperplasia (hematoxylin/eosin) and cartilage erosions (safranin O/fast green). (D) Histology scoring of paw joints of rats with PIA treated with vehicle (white) or 0.5 mg/kg IbTX (gray). Data are the mean ± S.E.M. (n = 4 paws per group). (E) Example X-rays of hind paws from vehicle-treated (left) and IbTX-treated (right) rats. (F) Ex vivo invasion of FLSs isolated from three healthy dark agouti rats, three rats with PIA treated with vehicle, and three rats with PIA treated with 0.5 mg/kg IbTX through Matrigel-coated Transwell inserts. Data are the mean ± S.E.M. (n = 3 per group). *P < 0.05; ***P < 0.001. Scale bar, 100 μm in (C).
Fig. 4.
Fig. 4.
(A) Clinical scores of paw inflammation from rats with PIA treated with vehicle (black) or with 0.5 mg/kg IbTX (gray) every other day starting 7 days after disease onset. Data are the mean ± S.E.M. (n = 5 to 6 rats per group). (B) Clinical scores of rats with PIA treated with vehicle (black) or with IbTX (gray) every other day for the first 7 days after disease onset. Data are the mean ± S.E.M. (n = 6 rats per group). *P < 0.05; **P < 0.01.
Fig. 5.
Fig. 5.
IbTX reduces disease severity in the CIA rat model of RA. (A) Clinical scores of paw inflammation of rats with CIA treated with vehicle (black) or 0.5 mg/kg IbTX (gray) every other day starting at disease onset. Data are the mean ± S.E.M. (n = 12 rats per group). (B) Ex vivo invasiveness of FLSs from three healthy Lewis rats, three rats with CIA treated with vehicle, and three rats with CIA treated with 0.5 mg/kg IbTX through Matrigel-coated Transwell inserts. Data are the mean ± S.E.M. (n = 3 per group). (C) Example X-ray images of paws from rats with CIA treated every other day with vehicle (left) or IbTX (right) for 14 days after disease onset. (D) Hematoxylin/eosin staining (top) and safranin O/fast green staining (bottom) of tissue sections of paws from rats with CIA treated with vehicle (left) or IbTX (right). Arrowheads indicate areas of hyperplasia (hematoxylin/eosin) and cartilage erosions (safranin O/fast green). (E) Histology scoring of paw joints of rats with CIA treated with vehicle (white) or IbTX (gray). Data are the mean ± S.E.M. (n = 3 paws per group). *P < 0.05; **P < 0.01. Scale bar, 100 μm in (D).
Fig. 6.
Fig. 6.
Paxilline, but not IbTX, causes tremors in rats. (A–C) Example acceleration recordings of rats treated with vehicle (A), 20 mg/kg paxilline (B), or 0.5 mg/kg IbTX (C). (D–F) RMS-transformed accelerations from the raw data presented in (A) to (C). Red lines indicate the threshold above background acceleration. (G) Numbers of tremor events above the threshold of the RMS-transformed acceleration of rats treated with vehicle, paxilline, or IbTX 10 minutes, 1 hour, and 24 hours after treatment. Data are the mean ± S.E.M. (n = 6 rats per group). (H) Tremor intensity of rats treated with vehicle, paxilline, or IbTX 10 minutes, 1 hour, and 24 hours after treatment. Data are the mean ± S.E.M. (n = 6 rats per group). *P < 0.05; **P < 0.01 compared with the vehicle-treated group.
Fig. 7.
Fig. 7.
Paxilline, but not IbTX, induces incontinence in rats. The number of urine spots counted from healthy Lewis rats treated with either vehicle, 20 mg/kg paxilline, or 0.5 mg/kg IbTX and then given an oral gavage of water and placed individually in a paper towel–lined cage for 1 hour. Data are the mean ± S.E.M. (n = 6 rats per group). **P < 0.01 compared with the vehicle-treated group.
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