Molecular Biophysics & Physiology, PhD - for continuing students only

Theoretical Descriptions of Membrane Ion Channels:

Robert S. Eisenberg works on the mechanisms of selectivity and permeation in ion channels. Ion channels are proteins with a hole down their middle that control a large fraction of the functions of life. Once open, spherical ions like sodium, potassium, calcium and chloride move through them by electro-diffusion. Dr. Eisensberg applies modern theories of ionic solutions to simple models of ion channels, using a variety of mathematical methods, from classical Metropolis Monte Carlo to modern methods of variational calculus, the energetic variational methods used in the theory of complex fluids. These simple models have been able to reproduce the selectivity properties of the calcium channels of the heart and of the sodium channels of nerve with the same set of parameters, using the (unchanging) crystal radii of ions. Current voltage relations are just now being calculated and there are signs that at least some of the properties of transporters and of (spontaneous) gating may emerge from models of this sort, without explicit structural changes.

Dirk Gillespie uses theories of liquids (like density functional theory, DFT) to model ion movement. All projects have a very close relationship with experimental groups that provide data and test the models’ predictions. Of particular interest are:

• Ryanodine receptor (RyR) and L-type calcium channels in muscle, which are involved in initiating muscle contraction, to understand how ions move through these pores (permeation) and why some kinds of ions are preferentially conducted (selectivity). The physiological consequences of these mechanisms (and their disruption in disease states) are also studied.
• Ca2+-induced Ca2+ release (CICR) in which the Ca2+ released by one RyR opens neighboring RyRs, who in turn open other RyRs. The goal is to understand the mechanism of CICR, the mechanism for stopping CICR, and changes in CICR during disease states
• Understanding and developing new nonbiological nanofluidic devices, devices that move electrolytes through nano-metersized pores in man made materials to create current. The goal is to understand their physics and to predict new unique device properties for future applications.

This lab is best suited for those with a background in physics and math who are interested in using, developing or implementing new modeling techniques, within an environment of collaboration with both theorists and experimentalists.

Gillespie, D. 2008. Energetics of divalent selectivity in a calcium channel: The ryanodine receptor case study. Biophys. J. 94:1169-1184.

Gillespie, D. and M. Fill. 2008. Intracellular calcium release channels mediate their own counter current: The ryanodine receptor case study. Biophys. J. 95:3706-3714.

He, Y., D. Gillespie, D. Boda, I. Vlassiouk, R.S. Eisenberg, and Z. S. Siwy. 2009. Tuning transport properties of nanofluidic devices with local charge inversion. J. Am. Chem. Soc. 131:5194-5202.

Proton Channels and NADPH oxidase. The main interest of Tom DeCoursey’s laboratory over the past decade has been in two molecules that reside in the membranes of white blood cells. These are proton channels and NADPH oxidase. Both play vital roles in white blood cells when these cells kill bacteria and other microbial invaders. When NADPH oxidase does not work, white cells cannot kill many types of bacteria. Patients afflicted with hereditary chronic granulomatous disease (CGD) lack this enzyme, and if not treated often die in childhood of recurrent infections. Dr. DeCoursey’s laboratory has shown that inhibiting proton channels prevents NADPH oxidase from working (DeCoursey et al, 2003, Nature 422:531-534) and that this results from the effects of proton channels on membrane potential and pH (Morgan et al, 2009, Proc. Natl. Acad. Sci., USA 106:18022-18027). They found that in human basophils, inhibiting proton channels prevents histamine release (Musset et al., 2008, Proc. Natl. Acad. Sci., USA 105:11020-11025). Others found that proton channels control sperm maturation. The DeCoursey laboratory continues to investigate roles played by proton channels in a variety of cells, such as B lymphocytes (Capasso et al, 2010, Nature Immunol. 11:265-272). They recently discovered a new proton channel gene in a dinoflagellate, which triggers the bioluminescent flash produced by these creatures when seawater is disturbed at night (Smith et al, 2011, Proc. Natl. Acad. Sci., USA 108:18162-18168).

Another focus of their current research is understanding how the proton channel works on a molecular scale. Because the proton channel gene was identified only in 2006, much work remains to be done. They design mutations to the protein, express the mutant channels in cultured cells and then record electrically from the cells to determine how the mutation affected the function of the molecule. For example, they found that the regulation of channel activity by phosphorylation occurs at a specific threonine residue in the intracellular part of the channel (Musset et al, 2010, J. Biol. Chem. 285:5117-5121). The figure shows how they believe the proton channel dimer is assembled (Musset et al, 2010, J. Physiol. 588:1435-1449). Very recently, this laboratory identified the “selectivity filter” of the human proton channel (the part that allows only H+ and no other ions to go through the channel) (Musset et al, 2011, Nature, 480:273-277).

Viral Fusion. Viruses deposit their genetic material into cells by fusing to membranes, initiating infection. Some viruses fuse directly to plasma membranes and others are internalized into endosomes where low pH triggers fusion. In both cases, the nucleocapsid leaves the viral interior by moving through the fusion pore into cytosol. Without fusion, the virion cannot infect the cell. There are three classes of viral fusion proteins. All types of viruses that utilize class II or class III proteins initiate infection by fusing from within endosomes. Fredric Cohen’s laboratory has found that fusion induced by class II and class III viral proteins (but not class I) is dependent on the voltage across the target membrane [refs]. If the voltage across an endosomal membrane was pharmacologically controlled, infection may be prevented. Furthermore, the similarity in structure of all class II proteins and the similarity between all class III proteins suggests that identifying a voltage sensor for one virus of a class should readily yield the sensor for many. The Cohen laboratory has also found that viral fusion mediated by class II or class III proteins varies with redox potentials, but fusion mediated by class I proteins do not. The range of values of redox potentials within interiors of endosomes differs for different types of endosomes. Variation of redox potentials can provide an important control for conformational changes of viral fusion proteins within endosomes. This has both conceptual and practical ramifications. Not all viruses within endosomes fuse and even a particular type of virus can enter cells through multiple endosomal pathways. The variation in redox potentials for endosomes of different pathways would account, at least in part, for variations in fusion and infectivity of virus within endosomes. This laboratory is pursuing the mechanisms by which membrane potentials and redox potentials regulate fusion and are characterizing how these two cellular controls of membrane fusion interact to guide fusion.

Membrane Cholesterol. Since so much of cellular biology in both health and disease is sensitive to cholesterol levels, experimentalists often measure cholesterol concentration under a wide variety of conditions. But from a physical point of view, the free energy of a system is the fundamental parameter that quantifies the tendency of a molecule to transfer from one state (or phase) to another. If each molecule is independent of all others, exhibiting zero interactions, the free energy is a function of the molecules’ concentrations. But for real materials, interactions between molecules alter the tendency to exchange between states. The free energy of transfer is then a function of the molecules’ “activity,” a thermodynamic parameter that accounts for interactions and that is an “effective concentration.” Determining activity allows one to infer the strength of molecular interactions. Although the difference between concentration and activity is a basic textbook distinction, a convenient and reliable method to measure cholesterol activity had not been developed. Fredric Cohen’s laboratory has now overcome this limitation, allowing us to thermodynamically characterize the dynamics of membrane cholesterol levels in living cells during physiological processes. This laboratory is currently investigating the changes in membrane cholesterol that occur during two important biological processes: cell proliferation stimulated by activation of the epidermal growth factor receptor (EGFR) and glucose uptake into cells as regulated by insulin. Their work demonstrates that activation of a signaling cascade leads to changes in cholesterol activity and that these changes in cholesterol activity negatively feedbacks on the signaling cascades, providing a mechanism for cellular homeostasis.

Regulation of Intracellular Calcium:

Eduardo Rios studies the workings of muscles, including skeletal and cardiac. The goal of this laboratory is to understand the cellular function of excitation-contraction (EC) coupling in terms of fundamental mechanisms. EC coupling translates electrical changes at the cell membrane to signals coded as increase in cytosolic [Ca2+], signals that result in muscle contraction. The mouse deploys extremely rapid calcium signals in its muscles, made possible by fast opening and closing of molecular channels that allow calcium to cross the walls of its cellular stores. In a working human, this gating is also very fast. But too much calcium may have unintended consequences, as it can become a signal that literally tells cells to start dying. So, the stability and health of muscle and other cells that “calcium-signal” is precarious, based on delicately tuned controls that must be as good at opening as closing channels to terminate the signal. The Rios laboratory has helped define the key molecular players in these functions. They use the mouse to study Ca2+ controls, comparing their operation and mechanisms in the healthy cell and in mice that have molecular abnormalities that copy human disease. The information and implications derived from these studies may apply to similar alterations in the heart, which lead to an irregular beat and may cause sudden death. Calcium signals also rule brain function, gut movements and blood pressure. This laboratory’s work has contributed and should continue contributing to the understanding of these functions and their diseases.

Thomas Shannon is interested in ionic channels, voltage gated ionic channels, fluorescence signal detection and electrophysiology, particularly as they relate to excitation-contraction coupling in striated muscle. Dr. 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. He 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 (i.e., sarcoplasmic reticulum Ca concentration) 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.

The research program led by Lothar A. Blatter focuses on the role of calcium ions for the regulation of cellular functions in the cardiovascular system. On the one hand they investigate, at the cellular and subcellular levels, through which pathways and mechanisms calcium itself is regulated in cellular compartments such as the cytosol, the nucleus, the sarcoplasmic reticulum and mitochondria. On the other hand they study how specific changes in cellular calcium concentrations control functions of cardiac myocytes and vascular endothelial cells. Of particular interests in cardiac cells are the regulation of calcium during excitation-contraction coupling, i.e., the rhythmic elevations of calcium that lead to contraction with every heartbeat, and in excitation-transcription coupling where this laboratory investigates the sources and specific roles of calcium for the activation of transcriptions factors (such as NFAT) that are involved in pathological cardiac remodeling. They are further interested in studying the specific changes in calcium signaling that occur in the diseased heart, i.e., in cardiac hypertrophy, heart failure and arrhythmias. In vascular endothelial cells we are interested in the interplay between calcium signaling and the generation of nitric oxide, an important endothelium-derived relaxing factor through which the vascular endothelium contributes to the control of blood flow and blood pressure. In both tissue types, an important area of research centers around the role of mitochondria for cardiovascular function. This research investigates the contribution of mitochondria to the regulation of cytosolic calcium concentration through its capability of storing and releasing calcium ions, but also the role of calcium ions for the regulation of mitochondrial functions, including energy metabolism and ATP production, regulation of mitochondrial channels such as the mitochondrial permeability transition pore, and control of the cellular redox state and protection against oxidative stress. For the study of these signaling processes and pathways this laboratory employs a wide palette of methodological approaches, ranging from high resolution confocal imaging using a large spectrum of fluorescent probes, to electrophysiology (patch clamp and lipid bilayer single channel recordings), photolysis of caged compounds, molecular biology and biochemical approaches, to the use of transgenic animals.

Michael Fill focuses on defining the mechanisms that control intracellular calcium signaling in excitable cells. They are particularly interested in the origin/control of local intracellular calcium signals generated by ryanodine receptor (RyR) channels. RyR channels are found in almost all cells and modulate cellular processes as diverse as contraction, secretion, synaptic transmission and transcription. However, most of this laboratory’s studies have focused on single RyR local control in mammalian striated muscle, where RyRs are found in abundance. Their research has delineated fundamental biophysical mechanisms important to normal or pathological muscle function. They generally apply a multiscale experimental approach that is best illustrated by their published works. Some recent works are listed below.

Qin et al. RyR luminal Ca regulation: Swapping calsequestrin and channel isoforms. Biophys. J. 97(7):1961-70, Oct. 7, 2009.

Liu et al. Flux regulation of cardiac RyR channels. J. Gen. Physiol. 135(1): 15-27, 2010.

Ramos-Franco et al. Ryanodol action on calcium sparks in ventricular myocytes. Pflugers Arch. 460(4):767-76, 2010.

Porta et al. Single RyR channel basis of caffeine’s action on Ca sparks. Biophys. J. 100(4):931-938, 2011.

Zhou et al. Carvedilol and its new analogs suppress arrhythmogenic store overload-induced Ca release. Nature Med. 17(8):1003-9, 2011.

Course Offerings

The following courses will be available, subject to demand and limitation, to students within The Graduate College:

• PHY-451 Physiology I
• PHY-452 Physiology II
• PHY - 501 Medical Physiology I Credit Hours: (4)
• PHY - 502 Medical Physiology II Credit Hours: (4)
• PHY - 503 Physiology of Striated Muscle Credit Hours: (3)
• PHY - 505 Introductory Membrane Biophysics Credit Hours: (3)
• PHY - 511 Graduate Physiology I Credit Hours: (4)
• PHY - 512 Graduate Physiology II Credit Hours: (4)
• PHY - 521 Mathematical Methods for Physiologists Credit Hours: (4)
• PHY - 524 Linear Differential Equations and Transform Methods Credit Hours: (3)
• PHY - 533 Programming-Numerical Methods Credit Hours: (4)
• PHY - 590 Special Topics in Physiology Credit Hours: (variable)
• PHY - 598 Introduction to Research Credit Hours: (variable)
• PHY - 640 Applied Electrophysiology Credit Hours: (4)
• PHY - 651 Advanced Topics in Muscle Physiology Credit Hours: (2)
• PHY - 690 Research Topics in Physiology Credit Hours: (variable)
• PHY - 699 Doctoral Research in Physiology Credit Hours: (variable)
• Molecular Biophysics & Physiology: Dissertation Process

Dissertation Proposal

Upon admission to the division, the student and his or her principal advisor will begin to make preparations for a proposal upon which the student’s original research project will be based. Such preparations will include intensive study of the literature in the student’s field of interest, instruction in the basic laboratory skills necessary for professional development in the field, and any other requirements established by the principal advisor and division director in addition to the course requirements discussed above. No later than 36 months after admission, the candidate will present to his or her dissertation committee an original proposal for contribution to knowledge in his or her area of specialization. It will include an extensive review of the relevant scientific literature, a description of the technical aspects of the proposed studies, an outline of the anticipated experimental approach to the major problem of interest and a discussion of possible results and their interpretation. The student will be expected to defend both his or her proposal and general ability to achieve professional competence before this committee. The dissertation committee will have at least three members: the principal advisor; the division director; and whenever possible, an individual outside the institution with national stature in the candidate’s field of interest, selected jointly by the candidate, Principal Advisor and Division Director. In addition to evaluating the content of the dissertation proposal, the outside member will have a responsibility to maintain close and frequent contact with the student and principal advisor and to advise the division director concerning the progress of the academic program. Ordinarily, the Dissertation Committee will be constituted as soon as possible after admission of a student to the division. The dissertation proposal may be submitted to the faculty prior to completion of course requirements in order to enable research activity to begin, but the student will not be formally admitted to candidacy until this is satisfactorily completed.

Candidacy

Upon acceptance of the dissertation proposal, the student will be admitted to doctoral candidacy and will be expected to devote fully his or her energies to the program. All students must meet a minimum residency requirement of one calendar year, following admission to candidacy, unless the Division Director and Dean grant special exceptions. The Principal Advisor will make frequent reports to the Division Director concerning the student’s progress. Should either faculty member or the candidate feel it appropriate, the Dissertation Committee can be called into session to judge the student’s continued participation in the graduate program or to determine possible alterations in the area of his or her research efforts. In addition, the student and Principal Advisor will be expected to consult periodically with the other committee members, who may also request the Division Director to call formal meetings of the Dissertation Committee. Conflicts between the student and/or any members of the Dissertation Committee not resolvable by the full committee may be referred to the Advisory Committee of the division or higher authority as specified in the policies and procedures of The Graduate College. The degree of Doctor of Philosophy is given in recognition of high attainment and ability in a particular field of scientific research as evidenced by submission of a dissertation showing power of independent investigation and forming an actual contribution to existing knowledge. Such dissertation will be submitted to the candidate’s dissertation committee for review and defended orally at least three months before the degree is granted. The dissertation committee will ordinarily request an evaluation of the candidate’s dissertation by a scientist of national stature not affiliated with Rush University. Acceptance of the dissertation by the dissertation committee will be reviewed by The Graduate College Council and the Dean, along with the candidate’s entire academic performance in The Graduate College. Determination of completion of all requirements will result in the Dean’s recommendation that the degree be awarded. Should the candidate not have submitted a dissertation three years after admission to candidacy, the dissertation committee will be convened to evaluate the candidate’s progress, and if proper, to suggest alteration in the program.