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

Abstract

Mouse B cells and their cell line (WEHI-231) express large-conductance background K(+) channels (LK(bg)) that are activated by arachidonic acids, characteristics similar to TREK-2. However, there is no evidence to identify the molecular nature of LK(bg); some properties of LK(bg) were partly different from the reported results of TREK type channels. In this study, we compared the properties of cloned TREK-2 and LK(bg) in terms of their sensitivities to ATP, phosphatidylinositol 4,5-bisphosphate (PIP(2)), intracellular pH (pH(i)), and membrane stretch. Similar to the previous findings of LK(bg), TREK-2 showed spontaneous activation after membrane excision (i-o patch) and were inhibited by MgATP or by PIP(2). The inhibition by MgATP was prevented by wortmannin, suggesting membrane-delimited regulation of TREKs by phosphoinositide (PI) kinase. The same was observed with the property of LK(bg); the activation of TREK-2 by membrane stretch was suppressed by U73122 (PLC inhibitor). As with the known properties of TREK-2, LK(bg) were activated by acidic pH(i) and inhibited by PKC activator. Finally, we confirmed the expression of TREK-2 in WEHI-231 by using RT-PCR and immunoblot analyses. The amplitude of background K(+) current and the TREK-2 expression in WEHI-231 were commonly decreased by genetic knockdown of TREK-2 using small interfering RNA. The downregulation of TREK-2 attenuated Ca(2+)-influx induced by arachidonic acid in WEHI-231. As a whole, these results strongly indicate that TREK-2 encodes LK(bg) in mouse B cells. We also newly suggest that the low activity of TREK-2 in intact cells is due to the inhibition by intrinsic PIP(2).

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.…”

“…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. However, a dramatic spontaneous increase of I TREK-2,w-c is observed with ATP-free pipette solution [27,36]. Based on the prevention of ATPinduced I TREK-2,i-o suppression by PI-kinase inhibitors (wortmannin or LY29402) and the inhibition by PI(4,5)P 2 [16,27,36], we proposed that TREK-2 inhibition by intracellular ATP is mediated by membrane-delimited production of PI(4,5)P 2 .…”

The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein

“…by antigens (5), cleavage fragments of the third complement component (6), and bacterial, viral, or autoimmunity host DNA (7), and even certain chemokines (8). PLC␥ 2 activation results in InsP 3 -mediated increases in the concentration of free Ca 2ϩ , diacylglycerol-mediated activation of protein kinases C, and changes in transmembrane signaling directly mediated by PtdInsP 2 (9).…”

B lymphocytes taken to task: a role for a background conductance K2P K+ channel in B cells. Focus on “Expression of TASK-2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells”

“…Further to this publication, the LK bg background K ϩ channel of WEHI-231 cells was identified as TREK-2 but a role in B cell function has not been advanced (23). Work with human T cells purified from peripheral blood has shown the presence of TWIK-related acid-sensitive K ϩ channel 1 (TASK-1) and TASK-3, acid-sensitive and G q protein-regulated K 2P channels.…”