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...
In this study, the short-circuit currents ( I(sc)) of electrolyte absorption and secretion in neonatal and adult rat colonic mucosa were compared and the role of Ca(2+) influx through luminal membranes examined in relation to the replenishment of intracellular Ca(2+) stores in colonic crypt cells. Neonatal tissues displayed higher amiloride-sensitive I(sc) and larger increases of electrogenic Cl(-) secretion in response to an increase in cytosolic [Ca(2+)] ([Ca(2+)](c)) or cAMP than found in adult colonic epithelium. Ca(2+)-mediated Cl(-) secretion as reflected in the I(sc) responses to carbachol ( I(sc,CCh)) showed milder "run-down" in neonates than in adult rats. We then employed the relatively stable I(sc,CCh) of the neonatal colon to investigate the polarity of Ca(2+) entry pathway after muscarinic stimulation. Repetitive stimulation with CCh under Ca(2+)-free conditions emptied the intracellular Ca(2+) stores and abolished the I(sc,CCh). Re-adding Ca(2+) to the basolateral perfusate rapidly restored I(sc,CCh) (about 71% of control in 10 min). In contrast, after re-adding Ca(2+) to the luminal perfusate only, the recovery of I(sc,CCh) took much longer and was incomplete, recovering to only 28% of control after 30 min. Recovery was accelerated by increasing [Ca(2+)] in the luminal perfusate (5 mM) and blocked by the presence of Gd(3+) (100 microM) in the luminal perfusate. The above results suggest that, in addition to the predominant role of Ca(2+) entry through the basolateral membrane, the influx of Ca(2+) through luminal membranes might also play a role in the Ca(2+) homeostasis of colonic epithelial cells.
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