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. 2016 May 10;18(1):103.
doi: 10.1186/s13075-016-1003-4.

Different expression of β subunits of the KCa1.1 channel by invasive and non-invasive human fibroblast-like synoviocytes

Zoltán Pethő  1   2 , Mark R Tanner  1   3 , Rajeev B Tajhya  1   4 , Redwan Huq  1   4 , Teresina Laragione  5 , Gyorgy Panyi  2 , Pércio S Gulko  5 , Christine Beeton  6   7   8
Affiliations

Different expression of β subunits of the KCa1.1 channel by invasive and non-invasive human fibroblast-like synoviocytes

Zoltán Pethő et al. Arthritis Res Ther. .

Erratum in

Abstract

Background: Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA-FLS) contribute to joint inflammation and damage characteristic of the disease. RA-FLS express KCa1.1 (BK, Slo1, MaxiK, KCNMA1) as their major plasma membrane potassium channel. Blocking KCa1.1 reduces the invasive phenotype of RA-FLS and attenuates disease severity in animal models of RA. This channel has therefore emerged as a promising therapeutic target in RA. However, the pore-forming α subunit of KCa1.1 is widely distributed in the body, and blocking it induces severe side effects, thus limiting its value as a therapeutic target. On the other hand, KCa1.1 channels can also contain different accessory subunits with restricted tissue distribution that regulate channel kinetics and pharmacology. Identification of the regulatory subunits of KCa1.1 expressed by RA-FLS may therefore provide the opportunity for generating a selective target for RA treatment.

Methods: Highly invasive RA-FLS were isolated from patients with RA, and FLS from patients with osteoarthritis (OA) were used as minimally invasive controls. The β subunit expression by FLS was assessed by quantitative reverse transcription polymerase chain reactions, Western blotting, and patch-clamp electrophysiology combined with pharmacological agents. FLS were sorted by flow cytometry on the basis of their CD44 expression level for comparison of their invasiveness and with their expression of KCa1.1 α and β subunits. β1 and β3 subunit expression was reduced with small interfering RNA (siRNA) to assess their specific role in KCa1.1α expression and function and in FLS invasiveness.

Results: We identified functional β1 and β3b regulatory subunits in RA-FLS. KCa1.1 β3b subunits were expressed by 70 % of the cells and were associated with highly invasive CD44(high) RA-FLS, whereas minimally invasive CD44(low) RA-FLS and OA-FLS expressed either β1 subunit. Furthermore, we found that silencing the β3 but not the β1 subunit with siRNA reduced KCa1.1 channel density at the plasma membrane of RA-FLS and inhibited RA-FLS invasiveness.

Conclusions: Our findings suggest the KCa1.1 channel composed of α and β3b subunits as an attractive target for the therapy of RA.

Keywords: Arthritis; Autoimmune disease; Cell migration; Electrophysiology; Patch clamp; Potassium channel; Regulatory subunit; Synovial fibroblast.

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Figures

Fig. 1
Fig. 1
Fibroblast-like synoviocytes from patients with rheumatoid arthritis express messenger RNA of multiple KCa1.1 β subunits. a expression fold measurements were conducted by quantitative reverse transcription polymerase chain reaction, compared with GAPDH expression (n = 6 donors with 3 independent repeats). Each bar shows expression of a different KCa1.1 subunit, the letters a–-e represent different transcript variants. b α/β ratio showing the amount of KCa1.1 α subunits expressed for each single β subunit. Note the different scales of the y-axis. ΔcT comparative cycle threshold, GAPDH glyceraldehyde 3-phosphate dehydrogenase
Fig. 2
Fig. 2
Fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS) express proteins of multiple calcium-activated potassium channel KCa1.1 β subunits. a Representative Western blot from a gel loaded with proteins from one RA-FLS donor with different lanes probed with antibodies against different subunits of KCa1.1 (top) and actin (bottom). b Intensity of KCa1.1 α and β subunit protein bands normalized to actin expression levels in RA-FLS. Each symbol on the scatterplot represents results from a different donor. The horizontal bar represents the mean for each subunit
Fig. 3
Fig. 3
Functional KCa1.1 β3b subunits are present on the plasma membrane of fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS). a Representative traces of whole-cell KCa1.1 currents elicited by 140-mV pulses for 200 milliseconds with 5 μM Ca2+ in the internal solution before (control) and after applying 2 μM paxilline (Pax), 100 nM charybdotoxin (ChTX), 30 μM arachidonic acid (AA), or 75 μM lithocholic acid (LCA). The two top traces are representative of ≥92 % of cells tested; the two middle traces are representative of approximately 70 % of cells tested; and the two bottom traces are representative of the other approximately 30 % of cells tested. b Peak KCa1.1 currents after different treatments normalized to the control levels. Mean ± SEM; n = 5 different donors. c Representative Western blot from a gel loaded with proteins from one RA-FLS donor or a rat testis extract. Different lanes were probed with antibodies against all splice variants of KCa1.1 β3 (pan-β3) or against KCa1.1 β3a, βc, βd, and βe only (top), and intensity of KCa1.1 β3 protein bands was normalized to actin expression levels in RA-FLS. Mean ± SEM; n = 6 different donors. d Activation kinetics (τAct) of RA-FLS K+ currents. Each symbol on the scatterplot represents a different cell. n = 5 different donors; **p ≤ 0.01, ***p ≤ 0.001
Fig. 4
Fig. 4
The calcium-activated potassium channel KCa1.1 β3 subunit is expressed by CD44high fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS), whereas the β1 subunit is expressed by CD44low RA-FLS and fibroblast-like synoviocytes from patients with osteoarthritis (OA-FLS). a Representative flow cytometry histograms showing expression of KCa1.1α by CD44high and CD44low RA-FLS. Gray shading represents control staining; black lines represent CD44 (left) or KCa1.1α (middle and right) staining. b Invasiveness of unsorted RA-FLS and RA-FLS from the same donors sorted by flow cytometry into CD44low and CD44high populations. Mean ± SEM; n = 3 RA-FLS donors. The line for the error bar for the control was thickened compared with other plots to make it visible. c Representative flow cytometry histograms showing expression of podoplanin, cadherin-11, and matrix metalloproteinase (MMP)-2 by CD44high (green) and CD44low (gray) RA-FLS. In the left panel, gray shading represents control staining and black lines represent CD44 staining. d Individual RA-FLS stained for CD44. The patch-clamp pipette’s shadow is visible on the right of each image. e K+ current density elicited at 140 mV in CD44high RA-FLS, CD44low RA-FLS, and OA-FLS. Mean ± SEM; n = 5 RA-FLS donors and 4 OA-FLS donors. f Activation kinetics (τAct) of K+ currents elicited at 140 mV in CD44high RA-FLS, CD44low RA-FLS, and OA-FLS. Mean ± SEM; n = 5 RA-FLS donors and 4 OA-FLS donors. g K+ current density after treatment of CD44high RA-FLS, CD44low RA-FLS, and OA-FLS with 75 μM lithocholic acid (LCA) or 30 μM arachidonic acid (AA) and normalized to current densities before treatment (horizontal dashed line). Mean ± SEM; n = 5 RA-FLS donors and 4 OA-FLS donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001
Fig. 5
Fig. 5
Silencing KCa1.1 β3 expression reduces cell surface expression of KCa1.1α. a K+ current density of untransfected fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS) and RA-FLS transfected with control small interfering RNA (siRNA) or with siRNA against KCa1.1 β3 before (black) and after treatment with 30 μM arachidonic acid (AA) (white; top plot) or 75 μM lithocholic acid (LCA) (gray; bottom plot) and normalized to current densities before treatment. Mean ± SEM; n = 4 RA-FLS donors. b Representative flow cytometry histograms showing background staining (gray shading), expression levels of KCa1.1α in untransfected RA-FLS (dashed line) and RA-FLS transfected with siRNA against KCa1.1β3 (solid black line; left histogram) or with control siRNA (solid black line; right histogram). The bar graph below shows the percentage of cells expressing KCa1.1α calculated from the flow cytometric profiles of three RA-FLS samples. Mean ± SEM. c K+ current densities of RA-FLS transfected with control siRNA (open boxes) or with siRNA against KCa1.1β3 (closed circles) and pulsed stepwise from −40 to 140 mV in 20-mV increments. Mean ± SEM; n = 4 RA-FLS donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001
Fig. 6
Fig. 6
Silencing calcium-activated potassium channel KCa1.1β3 but not KCa1.1β1 expression reduces inhibits the invasiveness of fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS). a K+ current density of untransfected RA-FLS and RA-FLS transfected with control small interfering RNA (siRNA) or with siRNA against KCa1.1 β1 before (black) and after treatment with 75 μM lithocholic acid (gray) and normalized to current densities before treatment. Mean ± SEM; n = 3 RA-FLS donors. b Invasiveness of untransfected RA-FLS and RA-FLS transfected with control siRNA, with siRNA against KCa1.1 β3, or with siRNA against KCa1.1 β1. Mean ± SEM; n = 4 RA-FLS donors. *p ≤ 0.5, **p ≤ 0.01, ***p ≤ 0.001
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