In WEHI-231, a representative immature B cell line, Ca2+ entry is paradoxically augmented by treatment with 2-aminoethoxydiphenyl borate (2-APB), a blocker of inositol 1,4,5-trisphosphate receptor and of nonselective cation channels (Nam, J. H., Yun, S. S., Kim, T. J., Uhm, D.-Y., and Kim, S. J. (2003) FEBS Lett. 535, 113-118). 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 IK,bg. The IK,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 BKbg and MKbg, respectively. The excision of membrane patches (inside-out (i-o) patches) greatly increased the Po of BKbg. In i-o patches, cytoplasmic MgATP (IC 50 = 0.18 mM) decreased the BKbg activity, although non-hydrolyzable adenosine 5′-(β,γ-imino)-triphosphate had no effect. A pretreatment with Al3+ or wortmannin (50 μM) blocked the inhibitory effects of MgATP. A direct application of phosphoinositide 4,5-bisphosphate (10 μM) inhibited the BKbg activity. Meanwhile, the activity of MKbg was unaffected by MgATP. In cell-attached conditions, the BKbg activity was largely increased by 2-APB. In i-o patches, however, the MgATP-induced inhibition of BKbg was weakly reversed by the addition of 2-APB. In summary, WEHI-231 cells express the unique background K+ channels. The BKbgs are inhibited by membrane-delimited elevation of phosphoinositide 4,5-bisphosphate. The activation of BKbg would hyperpolarize the membrane, which augments the calcium influx in WEHI-231 cells.