Cartilage-binding antibodies induce pain through immune complex-mediated activation of neurons.

Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.

The Role of Reactive Oxygen Species in ß-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome.

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute ß-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: ß-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to ß-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after ß-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute ß-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

Ion channel screening technology.

Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.

Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols.

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.

A biohybrid dynamic random access memory.

We report that GABA(A) receptors in a patch-clamped biological cell form a short-term memory circuit when integrated with a scanning-probe microfluidic device. Laminar patterns of receptor activators (agonists) provided by the microfluidic device define and periodically update the data input which is read and stored by the receptors as state distributions (based on intrinsic multistate kinetics). The memory is discharged over time and lasts for seconds to minutes depending on the input function. The function of the memory can be represented by an equivalent electronic circuit with striking similarity in function to a dynamic random access memory (DRAM) used in electronic computers. Multiplexed biohybrid memories may form the basis of large-scale integrated biocomputational/sensor devices with the curious ability to use chemical signals including odorants, neurotransmitters, chemical and biological warfare agents, and many more as input signals.

Microfluidic gradient-generating device for pharmacological profiling.

We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cell-based sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K+ channels (hERG) and ligand-gated GABA(A) receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC50 and EC50 values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.

A chemical waveform synthesizer.

Algorithms and methods were developed to synthesize complex chemical waveforms in open volumes by using a scanning-probe microfluidic platform. Time-dependent variations and oscillations of one or several chemical species around the scanning probe, such as formation of sine waves, damped oscillations, and generation of more complex patterns, are demonstrated. Furthermore, we show that intricate bursting and chaotic calcium oscillations found in biological microdomains can be reproduced and that a biological cell can be used as a probe to study receptor functionalities as a function of exposure to time-dependent variations of receptor activators and inhibitors. Thus, the method allows for studies of biologically important oscillatory reactions. More generally, the system allows for detailed studies of complex time-varying chemical and physical phenomena in solution or at solution/surface interfaces.

A microfluidics approach to the problem of creating separate solution environments accessible from macroscopic volumes.

We report on a microfluidic device that generates separate solution environments in macroscopic volumes. Spatially distinct patterns are created by emitting fluids from 16 different sources (closely spaced microchannels) into a solution-filled macroscopic chamber. The fluid in neighboring microchannels couples viscously in the macroscopic container, generating one single interdigitated stream. Scanning nanoelectrode amperometry was used for characterizing the concentration landscape and the diffusion zones between solutions running in parallel at different coordinates in the stream. These experiments were complemented by finite element simulations of the Navier-Stokes and mass transport equations to describe the velocity distributions and the diffusion behavior. For in channel flow velocities of 50 mm.s(-1), patterns could persist on the order of millimeters to centimeters in the open volume. The most narrow diffusion zones with widths less than 10 microm (5-95% concentration change) were found some tens of micrometers out in the macroscopic container. We demonstrate that a 14-microm-diameter nearly spherical object (biological cell) attached to a micropipet can be moved from one solution environment to another by a lateral displacement of only 8 microm. The device is suitable for applications where the solution environment around a microscopic or nanoscopic sensor needs to be changed multiple times, i.e., in order to build layered structures, for obtaining binding isotherms, and kinetic information, for example, on ion channels, enzymes, and receptors as well as in applications where different loci on an object need to be exposed to different environments or where complex solution environments need to be created for studies of interfacial chemistry between two streaming layers.

Nanotube-vesicle networks with functionalized membranes and interiors.

We describe nanotube-vesicle networks with reconstituted membrane protein from cells and with interior activity defined by an injection of microparticles or molecular probes. The functionality of a membrane protein after reconstitution was verified by single-channel ion conductance measurements in excised inside-out patches from the vesicle membranes. The distribution of protein, determined by fluorescence detection, in the network membrane was homogeneous and could diffuse via a nanotube connecting two vesicles. We also show how injecting small unilamellar protein-containing vesicles can differentiate the contents of individual containers in a network. The combination of membrane activity and interior activity was demonstrated by ionophore-assisted accumulation, and internal Calcium Green-mediated detection, of Ca2+ within a single network container. This system can model a variety of biological functions and complex biological multicompartment structures and might serve as a platform for constructing complex sensor and computational devices.

Stabilization of high-resistance seals in patch-clamp recordings by laminar flow.

The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.

A cell-based bar code reader for high-throughput screening of ion channel-ligand interactions.

This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10(3) ligand solutions in a single day.