Arachidonic acid-induced activation of large-conductance potassium channels and membrane hyperpolarization in mouse B cells

Abstract

Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we found that WEHI-231, an immature B cell line, expresses voltage-independent K(+) channels called large-conductance background K( + ) channels (LK(bg)). Arachidonic acid (AA) has attracted attention because of its potential regulatory roles in the apoptosis of immature B cells. To elucidate the functional targets of AA, we investigated the effects of AA on membrane currents, voltages, and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) of WEHI-231 and Bal-17 cells that represent immature and mature mouse B cells, respectively. In whole-cell patch clamp, both Kv and IKCa1 were inhibited by AA. On the other hand, AA activated LK(bg) current and non-selective cationic (NSC) current in WEHI-231 while only NSC current in Bal-17. Inside-out patch clamp study showed that AA directly activates LK(bg). AA induced hyperpolarization of WEHI-231 and depolarization of Bal-17 cells, respectively. The selective functional expression of LK(bg) and their activation by AA were also confirmed in the immature B cells (B220(+)/AA4.1(+)) freshly isolated from mouse spleen. In fura-2 spectrofluorimetry, AA induced persistent increase in [Ca(2+)](c) of WEHI-231 cells, which was attenuated by KCl-induced depolarization. In Bal-17 cell, however, AA induced only a transient increase of [Ca(2+)](c). In summary, the novel type of background K(+) channels (LK(bg)) in immature B cells is strongly activated while the other K(+) channels (Kv and IKCa1) commonly expressed in lymphocytes are inhibited by AA. The hyperpolarization and augmentation of Ca(2+) influx by LK(bg) activation might play a role in the response of immature B cells to AA.

Identification of the large-conductance background K+ channel in mouse B cells as TREK-2

“…The large conductance of LK bg in DVF condition is similar to the conductance of TREK-2 in the same DVF condition (8). Also, LK bg is activated by membrane stretch (23) and by unsaturated fatty acids (30).…”

“…Among them, TREK subfamily channels (TREK-1, TREK-2, and TRAAK) are relatively quiescent in basal states; they are strongly activated by physicochemical stimuli such as unsaturated fatty acids, membrane stretch, intracellular acidification, and heat (1,18,15). In contrast, the PKC-or PKA-dependent phoshorylation inhibits TREK-1 and -2 (8, 16).LK bg was the first characterized background K ϩ channel in immune cells and appeared to be preferentially expressed in immature B cells (22,30). Hence, clarifying the molecular identity of LK bg might be useful to understand B cell development.…”

“…A physiological role of LK bg was demonstrated from the augmentation of arachidonic acid-induced Ca 2ϩ influx by the concomitant activation of LK bg and strong hyperpolarization (30). Although the voltage independence of LK bg suggested an involvement of two-pore K ϩ channel (KCNK) family, the molecular identity of LK bg has been unsolved.…”

“…LK bg was the first characterized background K ϩ channel in immune cells and appeared to be preferentially expressed in immature B cells (22,30). Hence, clarifying the molecular identity of LK bg might be useful to understand B cell development.…”

Inhibition of TREK-2 K+ channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase

“…Previously, we reported that mouse primary B lymphocytes and B cell lines (WEHI-231) express a large-conductance K2P-like K + channel, which was identified as TREK-2 based on the following properties: (1) high levels of unitary conductance (>200 pS), (2) activation by intracellular acidification, (3) activation by polyunsaturated fatty acids (e.g., arachidonic acid), (4) activation by membrane stretch, and (5) inhibition by small interfering RNA against TREK-2 (siTREK-2) [26,27,35,36]. In B cells, despite the significant density of functional TREK-2 activities measured under inside-out (i-o) patch clamp recordings (I TREK-2,i-o ), the TREK-2 current measured by whole-cell (w-c) patch clamp (I TREK-2,w-c ) with ATPcontaining pipette solution is very small.…”