Two-pore domain potassium channel TREK-1 contributes to arachidonic acid-induced Ca2+ signaling in human fibroblast-like synovial cells

Figures

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  Fig. 1. Whole-cell patch-clamp recordings of quinidine-sensitive currents in hFLSs A. Representative superimposed whole-cell current traces in response to 500-ms voltage-command steps applied from holding potential of −40 mV to test potentials between +70 mV and −130 mV in control (a) and during exposure to 500 μM quinidine (b). Dotted line (red) indicates zero. B. Averaged current density-voltage (I–V) relationships of whole-cell currents recorded in control and during exposure to 500 μM quinidine. Inset: the I–V relationship of the quinidine-sensitive (difference) currents, which exhibits an outward rectification and a reversal potential around −63.3 ± 1.03 mV (n = 6), characterized by a Na+/K+ permeability ratio of approximately 0.05 when calculated using the Goldman-Hodgkin-Katz equation. hFLSs: Human fibroblast-like synovial cells.

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  Fig. 2. Functional expression of TREK-1 in hFLSs A. I–V relationships obtained by a voltage ramp in the absence (black) and presence (red) of DCPIB, and subsequent application of quinidine (blue). B. I–V relationships obtained by a voltage ramp in the absence (black) and presence (red) of ML402 and subsequent application of quinidine (blue). C and D. Representative immunofluorescence images for validation of synovial fibroblasts with CDH11 (green, C) and positive expression of TREK-1 (green, D). Nuclei (blue, C and D) were stained with Hoechst. Control (D) with a no-primary antibody.

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  Fig. 3. Modulation of TREK-1 channel by arachidonic acid (AA) in hFLSs A. Representative superimposed current traces in control (a) and during exposure to 10 μM AA (b). B. Averaged I–V relationships in control and during exposure to 10 μM AA. Inset: the I–V relationship of the AA-activated (difference) currents, exhibiting an outward rectification and a reversal potential approximately −56.9 mV. C. Concentration-dependent activation measured by percentage increase in the current amplitude at +70 mV before and after application of AA in the same cell. D. Effect of extracellular K+ concentration ([K+]o) on the reversal potential (Erev) of AA-activated currents. A fitting linear line shows a slope of 40.5 mV per 10-fold concentration change in [K+]o. E. Lack of the effect of AA in cells pretreated with spadin, a selective antagonist of the TREK1 channel.

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  Fig. 4. TREK-1 activation hyperpolarizes the resting membrane potential in hFLSs A. Typical example of the membrane potential recording in zero-current clamp mode. AA evoked hyperpolarization on the membrane potential. B and C. Averaged resting membrane potentials in control and 10 μM AA (B) or DCPIB (C). ∗p < 0.05 (n = 8), ∗∗p < 0.01 (n = 10).

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  Fig. 5. AA-induced intracellular Ca2+ mobilization in hFLSs A. Fluorescent images of intracellular Ca2+ signals in control (a) and after exposure to AA (b). hFLS cells loaded with Ca2+ fluorescent indicator Fluo8 B. (a) Averaged time courses of changes in relative fluorescent intensity (F/F0) of control cells (blue) and cells pretreated with spadin (Sp. orange) in the presence of extracellular Ca2+1.8 mM. (b) Averaged peak of F/F0, time to peak (Tpeak) and time to decay to 20 % of the peak (Tdecay). ∗p < 0.05, ∗∗p < 0.01. C. Experiments performed in extracellular medium with no added Ca2+.

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  Fig. 6. Cellular heterogeneity of Ca2+ responses to AA in hFLS A. Typical examples for four types of Ca2+ responses to AA: no response (a, type 1), a transient increase (b, type 2), sustained increase (c, type 3), and oscillatory increase (d, type 4). B. Percentage of the fraction of each cell type shown for control and in the presence of spadin, with or without 1.8 mM Ca2+ in the external medium.

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