Membrane-delimited Regulation of Novel Background K+ Channels by MgATP in Murine Immature B Cells

Abstract

The initial goal of the present study was to elucidate the effects of 2-APB on membrane currents, which revealed the presence of novel K ؉ channels in WEHI-231 cells. Under whole-cell patch clamp conditions, 2-APB induced background K ؉ current (I K,bg ) and hyperpolarization in WEHI-231 cells. Lowering of intracellular MgATP also induced the I K,bg . The I K,bg was blocked by micromolar concentrations of quinidine but not by tetraethylammonium. In a single channel study, two types of voltage-independent K ؉ channels were found with large (346 picosiemens) and medium conductance (112 picosiemens), named BK bg and MK bg , respectively. The excision of membrane patches (inside-out (i-o) patches) greatly increased the P o of BK bg . In i-o patches, cytoplasmic MgATP (IC 50 ‫؍‬ 0.18 mM) decreased the BK bg activity, although non-hydrolyzable adenosine 5-(␤,␥-imino)-triphosphate had no effect. A pretreatment with Al 3؉ or wortmannin (50 M) blocked the inhibitory effects of MgATP. A direct application of phosphoinositide 4,5-bisphosphate (10 M) inhibited the BK bg activity. Meanwhile, the activity of MK bg was unaffected by MgATP. In cell-attached conditions, the BK bg activity was largely increased by 2-APB. In i-o patches, however, the MgATPinduced inhibition of BK bg was weakly reversed by the addition of 2-APB. In summary, WEHI-231 cells express the unique background K ؉ channels. The BK bg s are inhibited by membrane-delimited elevation of phosphoinositide 4,5-bisphosphate. The activation of BK bg would hyperpolarize the membrane, which augments the calcium influx in WEHI-231 cells.Immune responses are initiated and regulated by the physical interactions of receptors and ligands on lymphocytes and antigen-presenting cells. In addition to the external factors, intrinsic changes of lymphocytes would participate in shaping the ongoing responsiveness of cells. An important intrinsic determinant of responsiveness could be the membrane potential (V m ) that is mainly determined by the potassium permeability of cell membrane and transmembrane gradient of [K ϩ ]. K ϩ channels, besides setting resting V m , play critical roles in various cellular functions (1). Among them, the modulation of calcium signals by providing electrical driving force has been suggested as an essential role of K ϩ channels (1, 2). In human primary T cells, two kinds of K ϩ channels, the voltage-gated K ϩ channels Kv1.3 and the calcium-activated K ϩ channel IKCa1 (hSK4), are found to play such a role; Kv1.3 channels are essential for activation of quiescent cells, and signaling through protein kinase C pathway enhances expression of IKCa1 channels that are required for proliferation (3, 4). Besides V m regulation, K ϩ channels also regulate the loss of intracellular K ϩ , which is a prerequisite step in the normotonic-or the Fas-mediated apoptosis (5, 6). In contrast to the studies in T cells, the characteristics of K ϩ channels and their roles in B cells have been rarely investigated, and the types of reported K ϩ channels are restricted to the v...

Effects of dietary or injected organic cations on larvalDrosophila melanogaster: Mortality and elimination of tetraethylammonium from the hemolymph

“…As discussed below, the synergism may result in part if one organic compound (e.g., quinidine) acts as a competitive inhibitor for the transport of another (e.g., TEA). Quinidine is also a K + channel blocker (Nam et al, 2004), so increased mortality in larvae maintained on diet containing both quinidine and TEA may be due to the combined effects of these compounds upon excretion of organic cations as well as actions upon K + channels.…”

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.…”

The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration

“…An important role of KCNK5 has been shown for so-called regulatory volume decrease, in which osmotocially swollen cells recover their volume by KCl loss through selective channels in different cell types [3,15,21]. Regulatory volume decrease pathways are often similar to apoptotic volume decrease, and in accordance, KCNK5 has been shown to induce apoptosis in kidney cells and B lymphocytes [41,54]. We here suggest that neurons in acute ischemic stroke start to express KCNK5, which exerts an important role in initiating the apoptotic cascade.…”