Protein Channels
Protein channels conduct ions through a narrow tunnel of fixed charge, thereby acting as gatekeepers for cells and cell compartments. Hundreds of types of channels are studied every day in thousands of laboratories because of their biological and medical importance: a substantial fraction of all drugs used by physicians act directly or indirectly on channels. Much of the work of Dr. Eisenberg and collaborators concerns open channels because a physical analysis of ion movement in an open channel should be easier than a physical analysis of most protein functions. Open channels have simple structure that does not change on the biological time scale and yet have important biological roles, e.g., in selecting one ion over another. A physical analysis of ion movement in an open channel should be easier than a physical analysis of most protein functions. The function of open channels can be described if the channel protein is described as an invariant arrangement of fixed electrical charges – not as an invariant set of rate constants or an invariant spatial distribution of potential. In that case, Duan Chen and Bob Eisenberg (and colleagues) have shown that the average electric field and current flow can be computed by the Poisson-Nernst-Planck (PNP) equations. The PNP equations describe the flux of ions (each moving randomly in the Langevin trajectories of Brownian motion) in the mean electric field specified in traditional (nonlinear) Gouy-Chapman/Debye-Hückel/Poisson-Boltzmann theories of electrolyte solutions and proteins at equilibrium. We have shown that PNP, in its one-dimensional version, fits a wide range of current voltage (I-V) relations – whether sublinear, linear or superlinear – from 6 types of channels, over ±180 mV of membrane potential, in symmetrical and asymmetrical solutions of 20 mM to 2 M salt. Using a three dimensional version of PNP, several laboratories are able to predict the I-V relations of the channel gramicidin within some 10 percent over a range of conditions, using the NMR structure, partial charges from standard molecular dynamics programs, and an estimate of the diffusion coefficient of Na+, made from separate data sets, using the one dimensional theory. The anomalous mole fraction (i.e., mixed alkali) effect in K+ channels and L-type calcium channels is easily explained. Single channels of porin and its mutants have been studied at length and our measurements of I-V relations are at least as extensive as measurements from any protein of known structure. Parameter estimates (in the mutants of known structure) are surprisingly close to those predicted (i.e., within 7 percent). Wolfgang Nonner, Dirk Gillespie, and Bob Eisenberg (and colleagues) have studied selectivity, the mechanism by which channels tell one ion from another. The dramatic selectivity of the L-type Ca channel of clinical fame arises naturally if the ions and glutamic oxygens of the selectivity filter of the channel are described as charged spheres, using the Mean Spherical Approximation or other representations of concentrated salt solutions. The fixed charge of the selectivity glutamates forces the channel to hold four positive mobile charges, making a concentration of some 17 molar univalent charge! Four (monovalent) sodium ions occupy twice the volume of two (divalent) calciums; the resulting difference in excluded volume produces calcium selectivity, by changing ion specific entropy and energy, and the overall electrostatic potential. This model predicts many of the selectivity properties of the channel in a wide range of ions and conditions after two adjustable parameters are set to optimal (unchanging) values. Taken together, these results suggest that open ionic channels are natural nanotubes—dominated by the enormous fixed charge lining their walls—in which atomic detail is unexpectedly unimportant because correlation effects are small. Highly charged nonequilibrium systems of this sort are hard to describe by direct simulations of molecular dynamics because those simulations are too small to represent a specific concentration of ions and too brief to compute flux or current. Traditional simulations also have difficulty with the electric field since they use periodic boundary conditions and equilibrium boundary conditions, at best.

Ionic Solutions
Dr. Eisenberg's work on ion channels has forced him to think a great deal about the properties of ionic solutions. These it turns out have been mostly studied in the tradition of equilibrium statistical mechanics or by direct simulations with atomic resolution in space and time (i.e., 0.1 Å and 1 femtosecond). Equilibrium analysis cannot predict the large currents that flow through channels. Direct simulations cannot describe systems large enough to have well defined concentrations, particularly of the trace ions and cofactors that control biological function. They cannot last long enough to reach the biological time scale. Zeev Schuss, Boaz Nadler and Bob Eisenberg are trying to develop an analysis of ions in solution based on the properties of their individual ionic trajectories. These can be described by a stochastic differential equation, which, in the ensemble, can satisfy nonequilibrium boundary conditions. The current observed through channel and the properties of ionic solutions arise from these trajectories. Because ions are charge and move rapidly (0.1 psec) in and out of small volumes (active sites of proteins, roughly a sphere of 14 Å diameter), the electrical potential varies a great deal (1 V) in short times and distances. The resulting forces cannot be ignored; indeed, they often dominate. Thus, the stochastic differential equations of motion of ions and water must be coupled to the equations of the electric field if they are to be realistic and Dr. Eisenberg and colleagues are trying to do that in a systematic manner.

Membrane Fusion
The goal of the laboratory of Fred Cohen and Grigory Melikyan is to understand membrane fusion. Membrane fusion is a complex protein-mediated process in which a fusion pore – the structure that joins two membranes and establishes continuity between two formerly separate aqueous compartments – forms and enlarges. Fusion between membranes is a key event in diverse cellular processes, including infection of cells. The proteins involved in viral binding and fusion have been unambiguously identified for many viruses.

Influenza Virus
More is known about the fusion of influenza virus induced by its envelope protein hemagglutinin (HA) than for any other protein. With Dr. Ruben Markosyan, Drs. Cohen and Melikyan are investigating the biophysical mechanism of fusion. They have shown that fusion occurs by first passing through the state of hemifusion. Hemifusion is a state in which contacting monolayer leaflets of a bilayer membrane merge prior to fusion pore formation. They have also shown that the transmembrane domain of HA is not absolutely required for fusion to occur, but greatly facilitates fusion and pore growth; they have identified and characterized intermediates of fusion and place them in chronological sequence; have showed that spontaneous curvature and other properties of lipids strongly affect pore growth. They are now determining how the approach of transmembrane domains toward fusion peptides induces fusion.

Human Immunodeficiency Virus (HIV)
Many viral fusion proteins, including those of influenza, HIV, SIV, Ebola, and respiratory syncytial virus, exhibit great structural similarity, folding into a rod-like structure of six-helix bundles. There is probably some common fusion mechanism conferred by the ability of proteins to fold into six-helix bundles. With Drs. Ruben Markosyan and Levon Abrahamyan, Drs. Melikyan and Cohen are using the fusion protein of HIV, Env, to investigate this mechanism. The bundles are known to be required for fusion to occur. This laboratory has shown that the bundles do not occur in the absence of membrane merger. This means that the bundle does not first form and then cause fusion. Rather a fusion pore is created by the movement of protein as it reconfigures into the six-helix bundle. This laboratory is now investigating whether the continuity of membranes created by formation of a pore allows additional Env to more readily fold into a six-helix bundle and thereby promote pore enlargement. Enlargement is necessary for release of the viral nucleocapsid into cytosol, a vital step in initiating infection.

Membrane Rafts
Lipid domains of membranes rich in cholesterol and sphingomyelin are the subject of great interest recently in cell biology because some important integral membrane proteins are preferentially located within them. Such domains are known as “rafts.” By studying these domains in model membrane systems, their physical chemistry can be isolated from a multitude of other processes that occur in biological membranes. Dr. Cohen's laboratory is using planar bilayer membranes and wide-field fluorescence microscopy to determine the basic physical-chemical properties of rafts. By using fluorescence probes that mark the rafts, they have shown that rafts are liquid-ordered phases and have characterized some of the viscoelastic properties.

Proton Channels
The properties and biological functions of ion channels are a long-term interest of Dr. Tom DeCoursey's laboratory. For many years potassium selective channels were studied in lymphocytes, macrophages, and lung epithelial cells. The main focus of the lab is now voltage-gated proton channels. In long collaboration with Dr. Vladimir V. Cherny, the fundamental properties of proton channels have been explored in alveolar epithelial cells. Modulation of the voltage-dependence of the channel by pHo and pHi ensures that it opens only when the electrochemical gradient for H+ is outward. In other words, when proton channels open, they extrude acid from cells, and acid extrusion is the main general function of these channels. A mechanism by which protons regulate the voltage-dependence of channel opening was proposed in collaboration with Vladislav Markin of the University of Texas at Dallas. The regulation of channel opening and closing by pH is hypothesized to occur by modulatory protonation sites, possibly histidine residues at either end of the channel molecule. Studies of the effects of temperature, H+ concentration, and deuterium on proton conduction through the channel suggest that proton channels are not water-filled pores like other ion channels. Instead, protons permeate by a Grotthuss-like mechanism, in which they hop across a hydrogen-bonded network through the channel protein. Dr. DeCoursey's colleagues (including Vladimir Cherny, Tatiana Iastrebova, Deri Morgan, Ricardo Murphy, and Larry L. Thomas from the Department of Immunology/Microbiology) also study proton channels in several types of white blood cells, including human neutrophils, eosinophils, and basophils. Recently, the single-channel conductance was determined in eosinophils in collaboration with Richard Levis (Rush) and Valerij Sokolov (Moscow). The single-channel currents (7-16 fA in amplitude) are 1000 times smaller than “normal” ion channel currents, and are the smallest unitary currents ever measured directly in any channel. In immune cells, the main function of proton channels is not pH regulation, but rather enabling NADPH oxidase to function.

NADPH Oxidase
When immune cells encounter bacteria or parasites, they secrete reactive oxygen species (superoxide, hydrogen peroxide, and bleach) that most consider to be lethal to pathogens. NADPH oxidase is the enzyme that produces these reactive oxygen species. NADPH oxidase is electrogenic because it works by translocating electrons across the cell membrane. Dr. DeCoursey's lab monitors the function of this enzyme in real time in living cells, by recording the electrical signal that it makes. Proton channels in immune cells serve to balance the electron transport by transporting an equal number of protons. This charge compensation is necessary, because electrical depolarization of the membrane potential by itself turns off the enzyme. Inhibiting proton current prevents NADPH oxidase function. Proton transport is measured electrically in the same cells at the same time. The ability to study electron and proton currents simultaneously in living cells as they respond to challenges has been a valuable tool to understand the relationships between these complex molecules. In collaborative studies with Dr. Larry L. Thomas of the Department of Immunology/Microbiology, the relationship between activation of NADPH oxidase and voltage-gated proton channels in phagocytes is under investigation. One useful approach has been use of cells with NADPH oxidase components genetically eliminated (“knock-outs”) or introduced by transfection. These studies are done in c ollaboration with Dr. Mary Dinauer of Indianapolis. The lab also studies naturally occurring knockouts, which exist in the leukocytes of patients with the rare hereditary chronic granulomatous disease.

Science Education
Research in the laboratory of Dr. Joel Michael continues to explore the learning and teaching of science (with a focus on physiology) at all post-secondary levels. Work is continuing on a “smart” computer tutor (for cardiovascular physiology) with natural language capabilities in collaboration with Dr. Martha Evens (Illinois Institute of Technology). Two studies are currently underway. One study is examining the learning outcomes that result from use the “smart” computer tutor (“CIRCSIM-Tutor”) and comparing these outcomes with the results of using a conventional computer-aided instructional program (“CIRCSIM”). A second study is examining the differences in tutoring behavior of novice and expert tutors with the goal of developing a set of the most effective tutoring rules. A second area of research is focused on the misconceptions that students bring to the classroom and approaches to helping them correct these faulty mental models. This work is being carried out in collaboration with Dr. Harold Modell (Physiology Educational Research Consortium, Seattle, WA) and Dr. Mary Pat Wenderoth (University of Washington). One study is aimed at determining the prevalence of misconceptions about cardiovascular, respiratory and renal physiology and whether some of these misconceptions arise from the students' inability to apply certain simple physical models to physiology. A second study is looking at the effects of different student laboratory protocols on remediating an existing respiratory misconception. Certain simple, easy to implement, changes to conventional laboratory protocols are demonstrating significant improvements in the “success” of these learning experiences.

Cellular signaling and the Regulation of Intracellular Calcium
The newly formed Section of Cellular Signaling is an environment of collaboration comprising laboratories and researchers interested in rapid signals that control metabolic changes within cells. The goals of Drs. Eduardo Rios and Jingsong Zhou's laboratory are to understand mechanisms that generate sudden changes in the concentration of the ion calcium inside cells. These changes control many metabolic responses and functions. Dr. Rios' group is especially interested in these events as they occur in muscles, including skeletal and cardiac muscle. In these tissues, calcium is stored inside the cell in the sarcoplasmic reticulum. Their work has helped determine the molecular makeup of the system that controls release of calcium from the sarcoplasmic reticulum into the cell. In collaboration with Gustavo Brum and Gonzalo Pizarro, of the University of Montevideo, his group has used an “ultrafast” confocal microscope to rapidly image events (Ca sparks) that constitute the minimal units or quanta of calcium release. These images revealed that the source of such sparks, hence the functional units of calcium release, are small groups or clusters of channels, which these researchers have named “couplons.” With Michael D. Stern and Heping Cheng, of the National Institute of Aging (Baltimore), Rios has used mathematical modeling techniques, to show that Ca sparks can be reproduced mathematically, assuming that channels within couplons are activated by the increase in cytoplasmic calcium concentration resulting from opening of individual channels within the group. In a collaborative project with Dr. Adom Gonzalez, Assistant Professor of Physiology, and Dr. Isaac Pessah (University of California at Davis), Dr. Rios used specific toxins, of both animal and plant origin. It was shown that these toxins may open one channel within a couplon, and that this opening often results in concerted opening of many other channels within the couplon, to produce a spark. This result confirmed the multi-channel origin of Ca sparks. Drs. Zhou and Rios seek to characterize the molecular interactions between calcium release channels, and dihydropyridine receptors, the voltage sensors of muscle. The studies start with the introduction of cDNAs of both molecules, in “wild type” form, or after mutations (structural changes), in cells usually devoid of these molecules. Production of these molecules by the foreign DNA helps understand how the changes in structure result in functional changes, hence providing insights to the relation between molecular structure and function. With Dr. Brad Launikonis, Dr. Zhou is investigating the determinants of changes in the release of calcium and frequency of Ca sparks during muscle development.

Viral Transduction of Foreign DNA into Muscle
An interesting recent advance by Dr. Zhou (in collaboration with Dr. Pompeo Volpe, University of Padova, Italy) is the development of techniques to introduce foreign DNA in adult cells of mammals and amphibians (i.e. generation of transgenic cells in living animals) using viral constructs (altered viruses containing the DNA of interest) that are injected into muscles of experimental animals. This technique is currently used to test the effects of overexpression of the calcium-storing protein calsequestrin.

Effects of Calcium Load on Cardiac Muscle
Also in the Section of Cellular Signaling, Dr. Thomas R. Shannon uses multiple biochemical and biophysical approaches to study the control of the load of calcium in the storage organelle (the sarcoplasmic reticulum) of normal and abnormal cells of the heart. Dr. Shannon has demonstrated on beating heart cells that the load in the normal sarcoplasmic reticulum is released partially to the cytosol in the process of a heart beat. Quantitative determination of these released fractions will allow him to understand the mutual interactions of Ca load and Ca release, and thus the control of contractile force, an important determinant of cardiac ejection (blood flow) in health and disease. For instance, Dr. Shannon has also demonstrated that the SR Ca load is reduced during heart failure and his research suggests that this reduction may be a critical factor in causing reduced cardiac contraction in this condition. Ongoing experiments are aimed at determining what causes this reduced SR Ca load.

Nonlinear Pattern Analysis
Much of physiological science concerns itself with the detection and analysis of “true signals” from out of the background of “noise.” The problem, however, is that 1) some signals often look like noise; or 2) other signals are heavily contaminated by noise. Dr. Joseph Zbilut in collaboration with Dr. Charles Webber, Jr. of Loyola University Medical Center, have been studying techniques that help elucidate these problems. This has evolved into the development of a technique called recurrence quantification analysis (RQA). With the help of Drs. Alessandro Giuliani (Italian Ministry of Health), Alfredo Colosimo and Cesare Manetti (both of the University of Rome “La Sapienza”) they have used this technique to gain insights into protein structure/function relationships. RQA has also been useful in understanding other complicated signal systems such as EEGs and ECGs. In collaboration with Dr. Mikhail Zak (Jet Propulsion Laboratory), Dr. Zbilut has developed an analysis of physiological dynamics which are characterized by singularities of equations of motion. Such equations can more realistically model constantly adapting systems-especially neural networks.