The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes

Abstract

Background and purpose: The ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis.

Experimental approach: Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells.

Key results: In cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons.

Conclusion and implications: Our results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.

Figure 1

Effect of DCPIB on currents activated by hypotonicity in primary cultured rat cortical astrocytes. A: Representative I/V traces of whole-cell currents evoked with a voltage protocol (inset) that from a holding potential of −40 mV hyperpolarizes the membrane potential to −120 mV for 3 s before the application of a slowly depolarizing ramp to +80 mV (40 mV·s⁻¹). Addition of 10 μM DCPIB decreased the hypotonicity-induced current at −120 mV but augmented the current magnitude at +80 mV. DCPIB effect was accompanied by a large hyperpolarization of current reversal potential. Upon washout (W.O.) with isotonic solution current returned to the basal level. B: Bar graph showing current densities assessed at −120 mV and +80 mV in isotonic solution (ISO) and under hypotonic conditions (HYPO) before and after 10 μM DCPIB application (n = 6). Statistical significance was calculated using paired two-tailed Student's t-test; *P < 0.05, **P < 0.01.

Figure 2

DCPIB activates a sustained K⁺-selective current in cultured astrocytes upon isotonicity. A: Representative I/V traces of astrocyte whole-cell currents evoked with the protocol in inset in standard bath solution (CTRL), following addition of 10 μM DCPIB (DCPIB) and after washout in standard bath solution (W.O.). DCPIB increased reversibly the outward current. B: Time-course of the percentage increase in current magnitude at +60 mV upon prolonged DCPIB application compared with the current before DCPIB administration (n = 11). C: Dose–response curve of the current increases following DCPIB challenge. Current densities of the DCPIB-induced outward currents measured at +60 mV were normalized to the control currents in the absence of DCPIB. The percentage increase in current densities fitted to a Hill equation yields a half-maximal concentration (EC₅₀) of 102 μM and Hill coefficient of 2.8. Numbers in brackets denote sample size (n). D: Representative I/V traces of whole-cell currents measured in standard 4 mM K⁺ bath solution in the absence (CTRL) and presence of 10 μM DCPIB (DCPIB) display an outwardly rectifying profile. In extracellular solution containing 100 mM K⁺ the DCPIB-evoked current reversed polarity at more positive membrane potentials and became linear suggesting the activation of open rectifier K⁺ channels. Currents returned outwardly rectifying after washout with the 4 mM K⁺ bath solution containing DCPIB (DCPIB W.O.). The voltage protocol is the same as in A.

Figure 3

DCPIB stimulates TREK-mediated K⁺ channels in cultured astrocytes. A–B: Representative I/V traces of control currents (CTRL) and currents evoked by DCPIB (10 μM) and AA (10 μM) in two different cells. Voltage stimulation as in Figure 2A. C: Comparison between ramp currents elicited by DCPIB (black) and AA (red). Each trace is the difference between evoked and control currents. Currents have been scaled in order to have the same amplitude at +60 mV. The result indicates that DCPIB activated a K⁺ conductance whose kinetics resembled those of the AA-induced current. D: Representative I/V traces of DCPIB-evoked currents in the absence and presence of AA (10 μM). Saturating concentration of DCPIB (200 μM) caused a large increase in K⁺ current which was not further augmented by co-application of AA. Voltage stimulation as in Figure 2A. E: Bar graph of K⁺ current densities assessed at −120 mV and +60 mV evoked in control conditions (CTRL) and after exposure to AA or DCPIB in the absence and presence of 500 μM quinidine. F: The same as in E but following application of 200 μM quinine. Column numbers denote sample size. Statistical significance was calculated using paired two-tailed Student's t-test; *P < 0.05 compared with controls (CTRL).

Figure 4

Cultured rat cortical astrocytes display immunosignals for K_2P 2.1 and K_2P 10.1 channel proteins A–C: Immunofluorescence staining of primary astrocyte cultures with antibodies directed against K_2P 2.1 (B) or K_2P 10.1 (C) channels and antibody against the cytosolic protein glial fibrillary acidic protein (GFAP). The two immunosignals do not overlay denoting that most of the K_2P 2.1 or K_2P 10.1 proteins are in the plasma membrane. In the absence of the primary antibodies (negat. ctrl) only GFAP was immunodetected (A). DAPI signals depict cell nuclei.

Figure 5

DCPIB but not the related molecule ethacrynic acid activates K_2P 2.1 and K_2P 10.1 channels expressed in transfected COS-7 cells. A: Representative I/V traces recorded in COS-7 cells transfected with K_2P 2.1 and the reporter gene EYFP (K_2P 2.1/EYFP) exposed to the standard bath solution (CTRL), after application of 10 μM DCPIB (DCPIB) and upon DCPIB washout with the standard bath solution (W.O.). B: Representative I/V traces of whole-cell recording in K_2P 10.1/EYFP-transfected cells in standard bath solution (CTRL), in the presence of 10 μM DCPIB and following washout (W.O.). C–D: Representative I/V traces of whole-cell recordings in K_2P 2.1/EYFP- (C) and K_2P 10.1/EYFP-transfected COS-7 (D) cells in standard bath solution (CTRL), after application of 30 μM EA, upon subsequent stimulation with 10 μM DCPIB, and following washout with standard bath solution (W.O.). For voltage stimulation protocol see Figure 2A.

Figure 6

Effect of DCPIB application on membrane properties of astrocytes and neurons in situ. A: Example of an astrocyte in the CA1 region of the hippocampus (stratum radiatum) in which recordings were obtained by the whole-cell patch-clamp method, filled with Alexa Fluor 488 hydrazide (AF488) and identified using an antibody directed against glial fibrillary acidic protein (GFAP). B: I/V plots of the membrane currents evoked in a hippocampal astrocyte by a voltage ramp protocol (see inset) before (CTRL) and after application of 100 μM DCPIB. C: DCPIB-evoked currents were measured in neurons in the granular cell layer of the dentate gyrus region in young rat hippocampal slices. D: Image of a neuron measured by the whole-cell patch-clamp method and filled with Alexa Fluor 488 hydrazide (AF488) during the measurement. E: I/V plots of the currents evoked in a dentate gyrus neuron by a slow ramp depolarizing the cell membrane from −100 mV to −20 mV for 2 s (see inset), before (CTRL) and after the application of 100 μM DCPIB. Note the background current increase after DCPIB application accompanied by a substantial hyperpolarization.

Figure 7

Immunolocalization of K_2P 2.1 and K_2P 10.1 proteins in hippocampal slices. A: Representative negative control (negat. ctrl) of immunostaining in the absence of primary antibodies against channel proteins. B: Immunohistochemical staining of fixed brain sections against K_2P 2.1 and glial fibrillary acidic protein (GFAP) in the CA1 region of the hippocampus. Arrowheads indicate positively-stained astrocytes. (s.p. – stratum pyramidale; s.r. – stratum radiatum) C: Staining for K_2P 2.1 and GFAP in the dentate gyrus. Arrowheads indicate positively-stained neurons. D: Immunohistochemical staining of fixed brain sections against K_2P 10.1 and GFAP in the CA1 region of the hippocampus. (s.p. – stratum pyramidale; s.r. – stratum radiatum) E: Staining for K_2P 10.1 channels and GFAP in the dentate gyrus. Arrowheads indicate positively stained neurons. DAPI signals in A–E depict cell nuclei.