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

Abstract

BackgroundFibroblast-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.MethodsHighly 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.ResultsWe 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 CD44high RA-FLS, whereas minimally invasive CD44low 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.ConclusionsOur findings suggest the KCa1.1 channel composed of α and β3b subunits as an attractive target for the therapy of RA.

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

“…Though KCa1.1 α subunits have a wide tissue distribution, many KCa1.1 channels are physically associated with regulatory β subunits that affect channel gating and activation and have restricted tissue distributions. Highly invasive, CD44 high , cadherin-11 high , and podoplanin high RA-FLS express KCa1.1 containing the β3 subunit (Pethő et al, 2016). In contrast, KCa1.1 α in the CNS is associated with the β4 subunit while the β1 subunit is expressed in smooth muscle cells.…”

“…The final version may differ from this version. Patch-clamp electrophysiology FLS were lifted with trypsin-EDTA, washed and patch-clamped using a Port-a-Patch automated system (Nanion Technologies), as described (Hu et al, 2012;Pethő et al, 2016;Tajhya et al, 2016).…”

“…In contrast, KCa1.1 α in the CNS is associated with the β4 subunit while the β1 subunit is expressed in smooth muscle cells. Because of the limited tissue distribution of KCa1.1 with the β3 subunit (Mannowetz et al, 2013;Pethő et al, 2016;Poulsen et al, 2009), it may be possible to utilize a blocker that affects KCa1.1 αβ3, but not KCa1.1 αβ4, as a step towards a novel therapy of RA that would not induce side effects in the CNS and as a proof of concept that targeting specific subunits of KCa1.1 can be used for tissuespecific pharmacology.…”

“…FLS from patients with RA (RA-FLS) and from rats with the pristane-induced arthritis (PIA) model of RA express the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) at their plasma membrane (Beeton, 2017;Hu et al, 2012;Pethő et al, 2016;Tanner et al, 2015). Inhibiting the expression or function of this channel reduces FLS invasiveness, proliferation, and protease secretion, while modulating cellular adhesion and integrin regulation (Hu et al, 2012;Tanner et al, 2015;Tanner et al, 2017a).…”

KCa1.1 channels as therapeutic targets for rheumatoid arthritis

“…RA-FLS are not homogeneous in terms of invasiveness as the most aggressive are highly invasive whereas the less aggressive RA-FLS and those isolated from the joints of patients with osteoarthritis (OA) are less invasive. We identified the type I transmembrane glycoprotein CD44 as a marker for aggressive human FLS 7 (Figure 1). An elevated surface expression of CD44 correlates with high levels of cadherin 11, podoplanin, and matrix metalloproteinase 2 (MMP-2) and with a highly invasive phenotype of human FLS.…”