Research in the Biomembrane Lab focuses on transport and signaling processes across biological membranes. The goal of the group is to increase the molecular understanding of these processes and ultimately to employ the ensuing insight to diagnose and possibly treat human disease. We are particularly interested in ion channel proteins that are involved in Alzheimer’s disease and in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and type I diabetes mellitus, as well as transporter proteins that are responsible for resistance to chemotherapy in treatment of cancer.

Figure: Panchika performing cell culture experiments.

Current research focuses on five main projects:

1. High-throughput profiling of ion channel activity in human lymphocytes as a biomarker for T cell-mediated autoimmune diseases
2. Chip-based transport assays of single multidrug resistance transporters
3. Screening for small molecules that inhibit neurotoxic ion channel activity by amyloid-beta (AB) peptides as a possible strategy to treat Alzheimer’s disease (AD)
4. Nanopore-based sensing of biomolecular assemblies and artificial ion channels
5. Interactions of proteins and therapeutic drugs with biomembranes and lipid rafts

Research in the Biomembrane Lab occurs in a multidisciplinary approach; we combine expertise in biochemistry, cell biology, and immunology with state-of-the art approaches in biophysics, electrophysiology, optical single molecule detection, and surface chemistry. The lab works in teams; current members include three postdoctoral researchers, six graduate students, and three undergraduate students.

1. High-throughput profiling of ion channel activity in human lymphocytes as a biomarker for T cell-mediated autoimmune diseases

Overview

Ion channels in cells of the immune system are promising targets for the treatment of human autoimmune diseases. In particular, the voltage-gated potassium channel, Kv1.3, which is expressed in human T cells, B cells, and natural killer cells, has emerged as a target for treatment of multiple sclerosis (MS), rheumatoid arthritis (RA), and type 1 diabetes. Despite the relevance of ion channels to human disease, current techniques to measure these ion channels such as patch clamp recordings are low-throughput, laborious, and require significant expertise. As a result, studying ion channels in cells of the immune system is not accessible to most clinicians and immunologists.

To address the challenges associated with studying ion channels in blood cells, we developed a high-throughput assay to measure Kv1.3 activity in immune cells isolated from human blood. This assay is automated, rapid, and able to measure Kv1.3 activity in 100-200 human T cells in less than one hour. This throughput is at least 20-fold higher than traditional techniques to measure ion channel activity. Moreover, this assay represents the first example of high-throughput profiling of ion channels using a primary cell type; the resulting methodology is general for other cell types and ion channel species.

Figure: General strategy for measuring Kv1.3 activity in blood cells. In an automated, high-throughput electrophysiology technology device, blood cells are seeded into a 384-well substrate, in which each well contains a small micropore. Upon application of suction, a single cell becomes attached to the micropore in each well, affording planar patch clamp measurements of ion current through voltage-gated channels in the attached cell. After initial recording of current, automated fluidics then add a drug compound that blocks the activity of Kv1.3 ion channels. Ion current is then measured again in the attached cells, and is greatly attenuated due to inhibition of Kv1.3 channels. The difference between currents before and after addition of compound is the resulting "Kv1.3-current", and this process proceeds for each of the 384 wells in an automated fashion.

To demonstrate the potential of this assay, we successfully profiled Kv1.3 activity in different lymphocyte subsets (e.g. CD4⁺ and CD8⁺ T cells, B cells, and gamma-delta cells) isolated from the blood of over 20 human subjects. We also studied the changes in Kv1.3 levels upon stimulation of human T cells to examine the use of Kv1.3 activity as a marker of T cell activation. Our results show that Kv1.3 activity correlated with upregulation of the IL-2 receptor (alpha chain) upon stimulation, and occurs before proliferation.

Figure: Cartoon depicting the timecourse of increase of Kv1.3 activity in human T cells upon stimulation, compared to other T cell activation markers. Data for Ca2+ were extrapolated from Lewis (Ann. Rev. Immunol., 2001, 19, 497-521); data for all other markers were obtained from average time-courses from four human subjects.

We are currently applying this novel assay to address relevant immunological and clinical questions. For instance, we are profiling Kv1.3 activity in T cells isolated from the blood of patients with MS and RA. As Kv1.3 activity is a sensitive marker of T cell activation, the high- throughput assay we developed may have clinical use for quantifying disease activity as well as for diagnosing and therapeutic monitoring of autoimmune disease.

2. Chip-based transport assays of single multidrug resistance transporters

Overview

Chemotherapy treatment often results in re-growth of cancer cells that are not only resistant to chemotherapeutic drugs, but are also resistant to other drugs (multidrug resistance, MDR). One of the major cellular processes associated with MDR is the expression of MDR-transporters on the plasma membrane of cancer cells. These MDR-transporters pump drugs out of cancer cells and thus reduce the effectiveness of chemotherapy treatments. In this NIH-funded project, we aim to develop novel assays to study transport (efflux rates) of single MDR-transporter proteins. Such assays combine several areas of expertise in our lab, including laser-machining of glass structures, planar patch clamp electrophysiology, formation of giant liposomes, and single-molecule fluorescence techniques. Ultimately, we hope that a better understanding of MDR transporter function and activation will accelerate the development of anti-cancer agents.

Figure: Schematic of a lipid bilayer sealed over a pore in a planar substrate. Prof. Mayer was a pioneer in developing this technique of planar electrophysiology, and we are currently using a similar setup, combined with single-molecule fluorescence microscopy techniques, to monitor transport through single MDR-transporters.

In terms of progress so far, we have recently published a manuscript on an optimized laser-based fabrication technique to generate microstructures for chip-based electrophysiology. Moreover, we have installed an optical single molecule detection technique called fluorescence correlation spectroscopy (FCS) in our lab and we have developed techniques to form giant liposomes in physiological solutions. We have recently carried out experiments of positioning these liposomes over micropores for planar lipid bilayer recordings.

Figure: Setup to form giant liposomes in a range of physiological solutions. Using the method of electroformation, surface-attached giant liposomes form in microfluidic chambers. These chambers allow the exchange of solution around the liposomes, and also inside the liposomes. Such a setup also allows monitoring the effects of the introduced solutions on interactions between neighboring liposomes. Click the following for sample movies of fusion of adjacent liposomes upon introduction of the following fusogenic agents: (Apple quicktime): Movie 1 (Ca²⁺-induced fusion), Movie 2 (PEG-induced fusion), Movie 3 (fusion by biospecific tethering)

Future work: We are in the process of adapting the FCS technique to the detection of the flux of fluorescent anticancer agents such as doxorubicin and rhodamine 123. Overall this research aims to develop assays that will make it possible to characterize better, and hence target effectively, multidrug resistance-associated transporters from cancer cells. The three specific aims of this R01 grant are:

Specific Aim 1: Develop chip-based functional transport assays for biophysical characterization of multidrug resistance-associated transporters. Combine these assays with electrophysiology and single molecule fluorescence detection.

Specific Aim 2: Investigate, under well-controlled conditions, the effect of parameters that can affect the transport rate, such as the transmembrane potential, the lipid composition, the concentration of ATP and co-transported molecules (e.g. glutathione) to understand better the function of MDR-associated transporters and possible ways to modulate this function.

Specific Aim 3: Develop assays that can monitor the transport rate of individual reconstituted MDR-associated transport proteins in planar lipid bilayers by taking advantage of extremely small volumes (picoliters) in microfabricated recording chips and by combining electrophysiology with single molecule optical detection. Use these assays to characterize MDR-associated transporters on the single-protein-level in analogy to single ion channel patch clamp recordings. Obtain single transporter efflux rates and investigate the activation state and the effect of activation (e.g. by phosphorylation) on the single transporter efflux rates.

3. Screening for small molecules that inhibit neurotoxic ion channel activity by amyloid-beta (AB) peptides as a possible strategy to treat Alzheimer’s disease (AD)

Overview

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder in the United States, affecting over 5 million people. AD is characterized by cognitive decline, memory loss, and a variety of neuropsychiatric symptoms and behavioral disturbances due to the death of brain cells. Currently, there is no cure that can delay or stop the deterioration of brain cells in AD. It is hence critically important to seek new strategies for treatment of this fatal neurodegenerative disease.

While there are several hypotheses for the pathogenesis of AD, a leading hypothesis involves deleterious effects of amyloid-beta (AB) peptides on neurons. One of the hallmarks of AD is the presence of AB plaques, which are formed by overproduction and aggregation of AB. In the biomembrane lab, we are interested in studying the interactions of AB peptides with biological membranes; more specifically, we are interested in screening small molecules that disrupt AB-membrane interactions as novel treatments for AD.

Figure: (Left) Cartoon illustrating plaques of amyloid-beta peptides around the neurons of patients with Alzheimer's disease. Image taken from www.ahaf.org/alzdis/about/AmyloidPlaques.htm. (Right) Image of fibrils of amyloid-beta peptides around neuronal cells in culture.

One approach we are employing to study the effects of AB peptides on biomembranes is to record the ion flux through planar lipid bilayers (PLB) treated with AB peptides. PLBs provide a well defined artificial biomembrane to detect changes in the membrane (e.g. formation of pores, thinning, or complete disruption) due to AB peptides. The formation of channels in membranes by AB peptides is especially interesting, as increased ion flux (especially of Ca²⁺) can lead to cell death. We have identified several compounds that inhibit increased transmembrane ion flux by AB peptides through PLBs.

Figure: Two examples of transmembrane ion flux through planar lipid bilayers induced by aggregated amyloid-beta peptides.

We are also investigating the effects of AB peptides on neuronal cells, examining both cell toxicity as well as electrical currents through cell membranes. For electrical recordings, we are performing whole-cell patch clamp (including studies with an automated electrophysiology technology, the Nanion Port-a-Patch) to screen for compounds that inhibit the increased transmembrane ion flux by AB peptides. Cell toxicity studies confirm the cytoprotective effects of these compounds that inhibit AB-induced ion flux.

Figure: Cell-based studies of the effects of AB peptides on cells. (A) Image of the Nanion automated electrophysiology device which affords whole-cell patch clamp measurements of transmembrane current in neuronal cells. (B) Cell toxicity of AB peptides at different concentrations and after different times of pre-incubation.

We have identified several promising compounds that both inhibit increased ion flux due to AB peptides and protect cells against AB peptides. Currently, we are testing these compounds in animal studies as a next step towards developing a potential novel therapeutic compound for treating AD.

4. Nanopore-based sensing of biomolecular assemblies and artificial ion channels

Overview

Sensitive and specific detection of biological agents in solution is of significant interest for a wide range of applications, including field-detection of biowarfare agents and diagnosis of human disease. We are currently using two novel methods for the development of nanopore-based sensors. The first method uses laser-fabricated nanopores to monitor, in situ, the formation of assemblies of nano-scale molecules or particles. The second method uses engineered ion channel-forming peptides to detect a chemical reaction on single molecules.

For the sensors based on nanofabricated pores in glass, we employ an electrical technique called "nano-Coulter counting" to detect molecules. In Coulter counting, an applied voltage drives an electrical current (from the flow of ions in solution) through a small pore. When a particle translocates through the pore, the resistance of the pore transiently increases, causing a decrease in the current through the pore. This decrease appears electrically as a "spike", with the magnitdue of the spike related to the size of the object passing through the pore. In "nano- Coulter counting", the pore consists of a submicron- or nano-pore (less than 1 um diameter), which allows detection of nanoscale particles and molecules. We use a custom laser fabrication technique to produce pores less than 500 nm diameter in low noise glass.

Figure: Schematic illustrating nano-Coulter counting. Application of a contant voltage drives ion current through a nanopore (which appears as a baseline electrical current with associate noise). The passage of particles through the pore increases the electrical resistance of the pore, creating what appears to be an electrical spike. The amplitude of the spike depends on the size of the particle relative to the size of the pore.

Using the principles of nano-Coulter counting, we were able to detect the presence of specific antibodies and viruses in solution. We were also able to detect the presence of staphylococcal-enterotoxin B (SEB), a potential bioterrorism agent, in serum. The advantages of the nano-Coulter counting technique are that it is label-free, sensitive, rapid, and can potentially be used in a cheap, disposable format for field detection of biological agents. Another advantage is that the technique makes it possible to follow the assembly of individual nanoscale objects (e.g. antibodies and antigen forming into immune complexes), an advantage that we are currently employing to study biological processes. Finally, we are interested in studying the theoretical aspects of nanopores, especially strategies to reduce electrical noise to improve the detection limits of nano-Coulter counting.

Figure: Monitoring the formation of nano-aggregates (in this case, immune complexes) using nano-Coulter counting. The addition of antibody to antigen (with multiple antibody-binding sites on the antigen) leads to the formation of immune complexes that grow larger over time. These immune complexes can be monitored by current deflections, which are proportional to the sizes of the complexes. Eventually, these immune complexes block the nanopore.

The second approach we are pursuing to design novel biosensors employs chemically modified ion channel-forming peptides. Gramicidin A (gA) is a naturally occuring peptide that can self-assemble into dimeric ion channel pores in biological membranes. A major advantage of gA is that it can be chemically modified to change the chemical group at the entrance of the ion channel channel pore. We found that the chemical change at the mouth of the pore can alter the conductance through the gA channel, allowing detection of the chemical reaction on a single gA pore -- and approach that we call "charge-based sensing".

Figure: Different conductances of gA molecules modified with different chemical groups at the opening of the gA pore.

In addition to detecting chemical reactions, we have also used chemically-modified gA molecules to detect enzyme activity in solution. Current work is focused on developing gA-based chemical sensors, as well as using gA as a biophysical model to study the relationship between chemical structure and ionic conductance of ion channels.

5. Interactions of proteins and therapeutic drugs with biomembranes and lipid rafts

Overview

Over 60% of pharmaceutical drugs target proteins or molecules in biological membranes; many of the remaining 40% must pass through biomembranes to exert effects on intracellular targets. Therefore, understanding the interactions between pharmaceutical drug compounds and proteins and lipids (including lipid rafts, which are emerging as highly relevant to human disease) is of significant interest. This project aims to establish new methods to screen for these interactions of proteins and pharmaceutical compounds with biomembranes of varying composition.

To establish such assays, we developed a technique to create arrays of lipid bilayers on glass substrates. This technique employed hydrogel stamps, in which tiny posts, each loaded with liposomes of a defined lipid composition, deliver lipids (forming a fluid bilayer) to a defined region of a glass chip. Among the benefits of hydrogel stamps is that the same lipid array can be delivered to over 100 individual glass chips without refilling of the posts. We used this technology to examine the targeting of several drugs to bilayers of different lipid composition. We have also extended this technology to stamp membranes with proteins, and also to stamp lipid raft regions.

Figure: Technique to form arrays of supported lipid bilayers on a glass substrate. A hydrogel stamp is first "inked" with solutions of liposomes, with a defined lipid composition for each individual post. The stamp is then pressed onto a glass substrate and quickly submerged in solution. Upon removing the stamp, an array of fluid lipid bilayers remains on the glass substrate in solution. The stamp can be used to create over 100 arrays before needing re-inking.

Figure: Quantifying the binding of fluorescently labeled protein to membrane arrays. Binding of the protein annexin V to membranes depends on Ca²⁺ and percentage of PS in membranes. Here, we stamped arrays of lipid bilayers with varying percentages of PS (from 0 to 50%) in two different CaCl₂ solutions (1 uM and 8 uM). We then quantified the fluorescence intensity of fluorescently-labeled annexin V bound to these membranes.

The ultimate goal for this research is to produce glass chips with tens to hundreds of functional lipid spots that contain the membrane composition of various human organs such as liver, kidney, ling, brain, etc. We envision using these chips for studies in the emerging field of lipidomics as well as for exploring the localization and distribution of pharmaceutical compounds in membranes of various composition. Finally, we will extend these membrane arrays to enable electrophysiological recordings in parallel using micropores below supported bilayers.