Mast cell stimulation by Ag is followed by the opening of Ca2+-activated K+ channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca2+-activated K+ channel KCa3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of KCa3.1 knockout mice (KCa3.1−/−) and their wild-type littermates (KCa3.1+/+). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcεRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in KCa3.1−/− BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca2+ ionophore ionomycin was followed by stimulation of Ca2+-activated K+ channels and cell membrane hyperpolarization in KCa3.1+/+, but not in KCa3.1−/− BMMCs. Upon Ag stimulation, Ca2+ entry but not Ca2+ release from intracellular stores was markedly impaired in KCa3.1−/− BMMCs. Similarly, Ca2+ entry upon endothelin-1 stimulation was significantly reduced in KCa3.1−/− cells. Ag-induced release of β-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in KCa3.1−/− BMMCs compared with KCa3.1+/+ BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in KCa3.1−/− cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by ∼50%) blunted in KCa3.1−/− mice. In conclusion, KCa3.1 is required for Ca2+-activated K+ channel activity and Ca2+-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.

Changes in the level of membrane cholesterol regulate a variety of signaling processes including those mediated by acylated signaling molecules that localize to lipid rafts. Recently several types of ion channels have been shown to have cholesterol-dependent activity and to localize to lipid rafts. In this study, we have investigated the role of cholesterol in the regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, activated transepithelial short circuit current (Isc), but only from the basolateral side. Similar results were obtained with a cholesterol-binding agent, filipin, and with the sphingomyelin-degrading enzyme, sphingomyelinase. Experiments with DeltaF508CFTR mutant mice indicated that raft disruption affected CFTR-mediated anion secretion, while pharmacological studies showed that this effect was due to activation of basolateral large conductance Ca2+-activated K+ (BK) channels. Sucrose density gradient centrifugation studies demonstrated that BK channels were normally present in the high-density fraction containing the detergent-insoluble cytoskeleton, and that following treatment with MbetaCD, BK channels redistributed into detergent-soluble fractions. Our evidence therefore implicates novel high-density cholesterol-enriched plasma membrane microdomains in the modulation of BK channel activation and anion secretion in colonic epithelia.

Listeriolysin, the secreted cytolysin of the facultative intracellular bacterium Listeria monocytogenes, is its major virulence factor. Previously, non-lytic concentrations of listeriolysin were shown to induce Ca²⁺-permeable nonselective cation channels in human embryonic kidney cells. In erythrocytes, Ca²⁺ entry is followed by activation of K⁺ channels resulting in K⁺-exit as well as by membrane scrambling resulting in phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. Phosphatidylserine exposure is a key event of eryptosis, the suicidal death of erythrocytes. The present study utilized patch-clamp technique, Fluo3-fluorescence, and annexin V-binding in FACS analysis to determine the effect of listeriolysin on cell membrane conductance, cytosolic free Ca²⁺ concentration, and phosphatidylserine exposure, respectively. Within 30 minutes, exposure of human peripheral blood erythrocytes to low concentrations of listeriolysin (which were non-hemolytic for the majority of cells) induced a Ca²⁺-permeable cation conductance in the erythrocyte cell membrane, increased cytosolic Ca²⁺ concentration, and triggered annexin V-binding. Increase of extracellular K⁺ concentration blunted, but did not prevent, listeriolysin-induced annexin V-binding. In conclusion, listeriolysin triggers suicidal death of erythrocytes, an effect at least partially due to depletion of intracellular K⁺. Listeriolysin induced suicidal erythrocyte death could well contribute to the pathophysiology of L. monocytogenes infection.

The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1−/−) and their wild-type littermates (sgk1+/+). The BMMC number as well as CD117, CD34, and FcεRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcεRI receptor, Ca2+ entry but not Ca2+ release from intracellular stores was markedly impaired in sgk1−/− BMMCs. The currents through Ca2+-activated K+ channels induced by Ag were significantly higher in sgk1+/+ BMMCs than in sgk1−/− BMMCs. Treatment with the Ca2+ ionophore ionomycin (1 μM) led to activation of the K+ channels in both genotypes, indicating that the Ca2+-activated K+ channels are similarly expressed and sensitive to activation by Ca2+ in sgk1+/+ and sgk1−/− BMMCs, and that blunted stimulation of Ca2+-activated K+ channels was secondary to decreased Ca2+ entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1−/− BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1−/− mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1−/− than in sgk1+/+mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.