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Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). In vitro, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol [...]

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both [[beta].sub.1]-adrenergic and [M.sub.2] muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is ~ 1.2 [micro]M, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both [[beta].sub.1] and [M.sub.2] receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though [[beta].sub.1] and [M.sub.2] receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors. [beta]-adrenergic receptor signaling; muscarinic receptor signaling; live cell imaging; fluorescence resonance energy transfer; biosensors

[Ni.sup.2+] inhibits current through calcium channels, in part by blocking the pore, but [Ni.sup.2+] may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of [ faster with 2 vs. 110 mM [Ca.sup.2+] or [Ba.sup.2+]. Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations (~3 [micro]M [Ca.sup.2+]). We conclude that both fast and slo 1 at a site outside the pore.

We examined the concentration dependence of currents through [Ca.sub.v]3.1 T-type calcium channels, varying [Ca.sup.2+] and [Ba.sup.2+] over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by [P.sub.Ca]/[P.sub.Na] = 87 and [P.sub.Ca]/[P.sub.Ba] = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent [[K].sub.d] values were similar for [Ca.sup.2+] and [Ba.sup.2+], both for block of currents carried by [Na.sup.+] (3 [micro]M for [Ca.sup.2+] vs. 4 [micro]M for [Ba.sup.2+], at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for [Ca.sup.2+] vs. 2.5 mM for [Ba.sup.2+]; nearly voltage independent). Block by 3-10 [micro]M [Ca.sup.2+] was time dependent, described by bimolecular kinetics with binding at ~3 x [10.sup.8] [M.sup.-1] [s.sup.-1] and voltage-dependent exit. [Ca.sup.2+.sub.o], [Ba.sup.2+.sub.o], and [Mg.sup.2+.sub.o] also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 [e.sup.-] per 98 [[Angstrom].sup.2] from Gouy-Chapman theory. Additionally, inward currents inactivated ~35% faster in [Ba.sup.2+.sub.o] (vs. [Ca.sup.2+.sub.o] or [Na.sup.2+.sub.o]). The accelerated inactivation in [Ba.sup.2+.sub.o] correlated with the transition from [Na.sup.+] to [Ba.sup.2+] permeation, suggesting that [Ba.sup.2+.sub.o] speeds inactivation by occupying the pore. We conclude that the selectivity of the 'surface charge' among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for [Ca.sup.2+] or [Ba.sup.2+].

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular 'pore gate' has also been proposed. We have used the voltage dependence of block by extracellular [Y.sup.3+] as a tool to locate the activation gate of the [alpha]1G ([Ca.sub.v]3.1) T-type calcium channel, [Y.sup.3+] block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by [Y.sup.3+] at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular [Ca.sup.2+] was replaced with [Ba.sup.2+], the rate of open block by [Y.sup.3+] was unaffected, but closed block was threefold faster than in [Ca.sup.2+], suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter. KEY WORDS: channel block * selectivity filter * patch clamp * voltage clamp * permeation

The [alpha]1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members ([alpha]1G and [alpha]1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat [alpha]1I stably expressed in HEK293 cells. We found several kinetic differences between [alpha]1G and [alpha]1I, including some properties that at first appear qualitatively different. Notably, [alpha]1I tail currents require two or even three exponentials, whereas [alpha]1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for [alpha]1I, even for relatively strong depolarizations. Despite these differences, gating of [alpha]1I can be described by the same kinetic scheme used for [alpha]1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 5--12-fold slower for [alpha]1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability. KEY WORDS: inactivation * activation * T-current * LVA channel * kinetic models

B.S. Rothberg and associate and Horrigan and associates both conducted experiments to study the mechanism of BK channel gating. They took very different approaches but arrived at compatible conclusions. While Rothberg et al examined in detail the gating of single BK channels under a limited range of conditions, Horrigan et al investigated macroscopic ionic and gating currents over a wide voltage range but in the effective absence of (Ca2+). Clearly, their goals were different, which explains why their models are based on what is known about channel structure.

The two K+ currents, M-current (I(sub M)) and delayed rectifier (I(sub DR)), in bullfrog sympathetic neurons have a permeability ratio of one and similar permeability sequences. Unlike I(sub M), I(sub DR) carries an inward Na+ current and its activity is suppressed by Na(sub o)(super +). Both channels carry a Cs+ current, and their activities are suppressed by the Cs+ current. I(sub M) shows no multi-ion pore characteristics while I(sub DR) does. The two K+ currents have dissimilar kinetics and modulation.

A study of the currents carried by Ba2+ ions through calcium channels in the sympathetic neurons of bullfrogs was conducted to show the effect of surface charges on saturated channels. The effect of surface charge on the saturation of channel with changing concentration and ionic strength of Ba2+ shows that the channel pore binds Ba2+ in a saturable way. The current-barium concentration relationship, however, may be considerably affected by surface charge. The channel permeation mechanism undergoes lesser surface charge than the gating mechanism.

The primary open-closed transition of single channel and whole-cell currents in the A7r5 cell line is voltage dependent as shown in the study of calcium channel gating kinetics. Dihydropyridine (DHP) agonists slow deactivation kinetics, shift the activation curve to more negative potentials, induce intermingled fast and slow channel openings and reduce the latency to the first opening. DHP agonists prove to be allosteric effectors that stabilize the open states of calcium channels in the Monod-Wyman-Changeaux model for channel activation.

Carbon nanofiber electrode architectures are used to provide for long-term, neuroelectroanalytical measurements of the dynamic processes of intercellular communication between excitable cells. Electroanalysis at discrete electrodes following long-term cell culture demonstrates that this platform remains responsive for the detection of easily oxidized species generated by the cultured cells.