Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.
Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells. However, the nature of the Ca(2+) sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca(2+) dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca(2+) dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca(2+) sensitivity of vesicle fusion at hair cell synapses.
ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X 1 (or P2X 3 ) receptors had strong desensitization, whereas currents in cells expressing P2X 2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X 1 receptor of either one of two 34-residue segments from the P2X 2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X 2 receptor only by providing both these segments of the P2X 1 (or P2X 3 ) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membranespanning segments. 2). A second difference is exemplified by the P2X receptor in the submandibular gland, where responses to ATP are not blocked by concentrations of the antagonists suramin and pyridoxalphosphate-6-azo-2,4-phenyldisulfonic acid (PPADS), which give complete block in smooth muscles or PC12 cells (3). The third difference is in desensitization; this is striking in some sensory neurons and in smooth muscles, where the current elicited by ATP declines in tens or hundreds of milliseconds, but in most other cells the current induced by ATP is sustained throughout applications lasting for several seconds (1).The differences in desensitization are reminiscent of those seen among subtypes of other ligand-gated ion channels, and might thus be expected to have important physiological sequela. For example, at glutamate-mediated synapses, desensitization can have a significant effect on synaptic transmission (4). The time constant of decay of synaptic currents mediated by ATP is in the range of 10-20 ms (5-7), suggesting that desensitization may play a role. Desensitization may also limit neurotoxicity. Those neurons that express slowly desensitizing glutamate receptors are more susceptible to excitotoxicity (8). Some P2X receptors have a high calcium permeability (see refs. 1 and 2); if this were coupled with slow desensitization, then cells would be more vulnerable to excessive calcium influx.Seven P2X receptor subunits have been cloned and expressed (refs. 9 and 10 and references therein). They belong to a structural class of channels distinct from the nicotinic superfamily (gated in vertebrates by acetylcholine, 5-hydroxytryptamine, ␥-aminobutyric acid, or glycine) and the glutamate family (gated by glutamic or aspartic acid). Each channel subunit appears to have only two transmembrane segments and a large, extracellular loop (see refs.
Background and purpose: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). Experimental approach: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. Key results: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 mM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by abmethylene ATP (10 mM, 94% cells, n = 31) and was antagonized in LAD2 cells by NF 449 (1 mM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 mM). A P2X7-like nondesensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n = 24; 54% HLMCs, n = 24) which was antagonized by AZ11645373 (1 mM). P2X7-like responses were also evoked in LAD2 cells by 2′(3′)-0-(4-benzoylbenzoyl)ATP (300 mM). A P2X4-like current was evoked by 100 mM ATP (80% LAD2, n = 10; 21% HLMCs, n = 29), the amplitude and duration of which was potentiated by ivermectin (3 mM).
Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca2+ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg2+ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family. In this study, we have investigated TRPM7 expression and function in primary human lung mast cells (HLMCs) and in the human mast cell lines LAD2 and HMC-1, using RT-PCR, patch clamp electrophysiology, and RNA interference. Whole cell voltage-clamp recordings revealed a nonselective cation current that activated spontaneously following loss of intracellular Mg2+. The current had a nonlinear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reducing external divalent concentration from 3 to 0.3 mM dramatically increased the size of the outward current, whereas the current was markedly inhibited by elevated intracellular Mg2+ (6 mM). Ion substitution experiments revealed cation selectivity and Ca2+ permeability. RT-PCR confirmed the presence of mRNA for TRPM7 in HLMC, LAD2, and HMC-1 cells. Adenoviral-mediated knockdown of TRPM7 in HLMC with short hairpin RNA and in HMC-1 with short interfering RNA markedly reduced TRPM7 currents and induced cell death, an effect that was not rescued by raising extracellular Mg2+. In summary, HLMC and human mast cell lines express the nonselective cation channel TRPM7 whose presence is essential for cell survival.
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