Peripherally induced human regulatory T cells uncouple Kv1.3 activation from TCR-associated signaling.

Peripherally induced Tregs (iTregs) are being recognized as a functional and physiologically relevant T-cell subset. Understanding the molecular basis of their development is a necessary step before the therapeutic potential of iTreg manipulation can be exploited. In this study, we report that the differentiation of primary human T cells to suppressor iTregs involves the relocation of key proximal TCR signaling elements to the highly active IL-2-Receptor (IL-2-R) pathway. In addition to the recruitment of lymphocyte-specific protein tyrosine kinase (Lck) to the IL-2-R complex, we identified the dissociation of the voltage-gated K(+) channel Kv1.3 from the TCR pathway and its functional coupling to the IL-2-R. The regulatory switch of Kv1.3 activity in iTregs may constitute an important contributing factor in the signaling rewiring associated with the development of peripheral human iTregs and sheds new light upon the reciprocal crosstalk between the TCR and the IL-2-R pathways.

A semi-synthetic ion channel platform for detection of phosphatase and protease activity.

Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP). Enzymatic hydrolysis of this phosphate group removes negative charges from the entrance of the gA pore, resulting in a product with measurably reduced single ion channel conductance compared to the original gA-phosphate substrate. This technique employs a standard, commercial bilayer setup and takes advantage of the catalytic turnover of enzymes and the amplification characteristics of ion flux through individual gA pores to detect picomolar concentrations of active AP in solution. Furthermore, this technique makes it possible to study the kinetics of an enzyme and provides an estimate for the observed rate constant (k(cat)) and the Michaelis constant (K(M)) by following the conversion of the gA-phosphate substrate to product over time in the presence of different concentrations of AP. In the second case, modification of gA with a substrate for proteolytic cleavage by anthrax lethal factor (LF) afforded a sensitive method for detection of LF activity, illustrating the utility of ion channel-based sensing for detection of a potential biowarfare agent. This ion channel-based platform represents a powerful, novel approach to monitor the activity of femtomoles to picomoles of two different classes of enzymes in solution. Furthermore, this platform has the potential for realizing miniaturized, cost-effective bioanalytical assays that complement currently established assays.

Films of agarose enable rapid formation of giant liposomes in solutions of physiologic ionic strength.

This paper describes a method to form giant liposomes in solutions of physiologic ionic strength, such as phosphate buffered saline (PBS) or 150 mM KCl. Formation of these cell-sized liposomes proceeded from hybrid films of partially dried agarose and lipids. Hydrating the films of agarose and lipids in aqueous salt solutions resulted in swelling and partial dissolution of the hybrid films and in concomitant rapid formation of giant liposomes in high yield. This method did not require the presence of an electric field or specialized lipids; it generated giant liposomes from pure phosphatidylcholine lipids or from lipid mixtures that contained cholesterol or negatively charged lipids. Hybrid films of agarose and lipids even enabled the formation of giant liposomes in PBS from lipid compositions that are typically problematic for liposome formation, such as pure phosphatidylserine, pure phosphatidylglycerol, and asolectin. This paper discusses biophysical aspects of the formation of giant liposomes from hybrid films of agarose and lipids in comparison to established methods and shows that gentle hydration of hybrid films of agarose and lipids is a simple, rapid, and reproducible procedure to generate giant liposomes of various lipid compositions in solutions of physiologic ionic strength without the need for specialized equipment.

High-throughput profiling of ion channel activity in primary human lymphocytes.

We present a high-throughput method to quantify the functional activity of potassium (K (+)) ion channels in primary human lymphocytes. This method is rapid, automated, specific (here for the voltage-gated Kv1.3 ion channel), and capable of measuring, in parallel, the electrical currents of over 200 individual lymphocytes isolated from freshly drawn blood. The statistics afforded by high-throughput measurements allowed direct comparison of Kv1.3 activity in different subsets of lymphocytes, including CD4 (+) and CD8 (+) T cells, gammadelta T cells, and B cells. High-throughput measurements made it possible to quantify the heterogeneous, functional response of Kv1.3 ion channel activity upon stimulation of CD4 (+) and CD8 (+) T cells with mitogen. These experiments enabled elucidation of time-courses of functional Kv1.3 activity upon stimulation as well as studies of the effects of the concentration of mitogenic antibodies on Kv1.3 levels. The results presented here suggest that Kv1.3 ion channel activity can be used as a functional activation marker in T cells and that it correlates to cell size and levels of a surface antigen, CD25. Moreover, this work presents an enabling methodology that can be applied widely, allowing high-throughput screening of specific voltage-gated ion channels in a variety of primary cells.

Triggering and visualizing the aggregation and fusion of lipid membranes in microfluidic chambers.

We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an approximately 6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions.

Giant liposomes in physiological buffer using electroformation in a flow chamber.

We describe a method to obtain giant liposomes (diameter 10-100 microm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.

Electroformation of giant liposomes from spin-coated films of lipids.

This paper describes spin-coating of solutions of lipids and using the resulting thin films for electroformation of giant liposomes. Spin-coating made it possible to generate uniform films of lipids with controllable thickness over large surfaces (>25 cm(2)) of indium tin oxide. Establishing a range of thicknesses optimal for electroformation (25-50 nm), we demonstrate formation of giant liposomes from lipids (such as asolectin, phosphatidylserine, and phosphatidylglycerol) that do not readily form giant liposomes from traditional, droplet-derived films. We compared liposomes from a spin-coated film of lipids to liposomes formed from traditional droplet-derived films and found that spin-coated films produced larger (by factor of 2-5) and more abundant liposomes than droplet-derived films of lipids. Electroformation from spin-coated, homogenous lipid films of optimal thickness provided a reproducible way to obtain liposomes with diameters that are predominantly larger than 30 microm over the entire surface of formation.