Olaf S. Andersen, M.D. - US grants

The mechanisms of ion movement through lipid bilayers will be studied with emphasis on ion transport through, and ion selectivity of, polypeptide channels incorporated into the bilayer. Specifically the behavior of channels formed by gramicidin A will be investigated in detail. Ion movement through gramicidin A channels in diphytanoylphosphatidylcholine membranes will be studied for the alkali metal cations and other monovalent cations using single-channel current-voltage characteristics, ion tracer fluxes, as functions of the aqueous activity of the permeant ion, as well as the equilibrium binding of the ion to the channel as the primary experiments. These data will be used to develop a model for ion movement through gramicidin A channels. It should then be possible to predict the ion-selectivity properties of the channels, these predictions will be tested by biionic potential measurements as well as single-channel conductance measurements in symmetrical mixtures of two permeant ions. Serious discrepancies between predictions and experiment will be investigated further to characterize their molecular basis, in particular to see if ion-induced structural changes can occur. Other experiments will be concerned with the molecular behavior of the gramicidin A channels, the kinetics of channel formation and channel dissociation will be studied as functions of the type and concentration of the permeant ions, to see to what extent permeating ions can influence the channel behavior on a rather gross level. Experiments with different gramicidin A analogs, and with membranes of different lipid composition will be used to obtain a clearer picture of the molecular details of ion movement through these channels. The information thus obtained will be used to illustrate principles of ion movement through single-filing channels. They will also be used to elucidate how phloretin, a powerful membrane-modifier, will affect a transmembrane channel.

The long-term goal of the proposed research is to understand the molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on low-molecular channel formers, the linear gramicidins where structural information is available, and through studies on macromolecular channels, the voltage-dependent sodium channels. The kinetics of ion movement through gramicidin channels will be studied to delineate the steps involved in ion entry and ion exit. The major aim is to provide a dissection of the ion entry step. Linear gramicidins which have been site-specifically modified will be used to obtain a structure-function relation for a transmembrane channel. The emphasis will be on gramicidins where the polarity of the side chains are systematically varied at their N- and C-terminal ends, where the length is varied (from 13 to 17 amino acids), or where there are fixed negative charges at the channel entrances. The single-channel conductances and lifetimes will be studied as a function of permeant ion type and concentration, and the structural equivalence of the different compounds will be examined based on the formation of hybrid channels between the modified peptides and reference compounds, e.g. valine gramicidin A. The modulation of the channel behavior by extrinsic factors, such as changes in interfacial dipole potentials, will be studied. Voltage-dependent sodium channels from mammalian forebrain and cardiac muscle will be studied to get insight into the mechanism of ion entry into the channels, especially whether negative surface charges close to the channel entrance have a physiological function as guides for ion entry. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the TTX binding site and the extracellular channel entrance. These studies will also contribute to a better understanding of the mechanism of neurotoxin (STX and TTX) induced block. The stationary voltage-activating characteristics will be examined, to characterize the slow "state changes" which affect the gating (shift the midpoint of activation curves).

The goal of the proposed research is to understand some of the fundamental properties of mammalian presynaptic nerve terminals. Pinched-off nerve endings (synaptosomes) isolated from rat brain will be used, because they retain many functional, metabolic, and morphological properties of intact neuronal tissue, and are well-suited for studies that involve radiotracer-flux, optical, and biochemical techniques. The Investigator will: 1) examine the characteristics of the presynaptic Ca channels involved in neurosecretion - permeability and selectivity, and mechanism of inactivation; 2) determine how cations that permeate, and selectively block, Ca channels influence Ca sequestration and extrusion and transmitter release; 3) measure intracellular pH in the nerve terminals, study how it is regulated, and how it is related to Ca channel activity and neurosecretion; 4) develop and utilize methods for loading Ca-indicators into nerve terminals, so that Ca metabolism and its relation to the activity of nerve terminals can be investigated. This project will provide a better understanding of the basic cellular mechanisms that control intracellular Ca and pH in brain nerve endings, and of how this regulation affects the relase of brain neurotransmitters.

The long-term goal of the proposed research is to understand molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on voltage-dependent sodium channels that are incorporated into planar lipid bilayers of defined composition. Ion permeation, pharmacological modification, and voltage activation (gating), will be examined, with special emphasis on the role of fixed charges at the extra- and intracellular surface of the protein and host bilayer in modulating channel function. The mechanism of ion entry into the channels will be studied to determine whether negative charges close to the channel entrance have a physiological function as guides for ion entry. Ion permeability and block of channels modified by batrachotoxin, veratradine, pyrethroid insecticides, and group-specific modification will be compared in an attempt to clarify why channels that have a decreased single-channel conductance have a decreased ion selectivity. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the guanidinium toxin binding site and the extracellular channel entrance. The kinetics of toxin-induced channel closures will be examined to determine whether the guanidinium toxin-induced channel closures occur as a two-step event, where the channel is closed thorough a conformational change subsequent to toxin binding. The stationary voltage-activation of single channels will be examined to define to what extent gating behavior is affected by lipid surface charges, and to further define the asymmetry in the apparent surface charge density at the extra- and intracellular surfaces of the channel. The slow "mode changes" that affect gating will be studied. The aim is to characterize some of the stationary conformational fluctuations that occur in an integral membrane protein, as well as to determine whether the mode changes can be fully accounted for by discrete shifts in the midpoint potential of the activation curves.

The long-term goal of the proposed research is to understand molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on voltage-dependent sodium channels that are incorporated into planar lipid bilayers of defined composition. Ion permeation, pharmacological modification, and voltage activation (gating), will be examined, with special emphasis on the role of fixed charges at the extra- and intracellular surface of the protein and host bilayer in modulating channel function. The mechanism of ion entry into the channels will be studied to determine whether negative charges close to the channel entrance have a physiological function as guides for ion entry. Ion permeability and block of channels modified by batrachotoxin, veratradine, pyrethroid insecticides, and group-specific modification will be compared in an attempt to clarify why channels that have a decreased single-channel conductance have a decreased ion selectivity. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the guanidinium toxin binding site and the extracellular channel entrance. The kinetics of toxin-induced channel closures will be examined to determine whether the guanidinium toxin-induced channel closures occur as a two-step event, where the channel is closed thorough a conformational change subsequent to toxin binding. The stationary voltage-activation of single channels will be examined to define to what extent gating behavior is affected by lipid surface charges, and to further define the asymmetry in the apparent surface charge density at the extra- and intracellular surfaces of the channel. The slow "mode changes" that affect gating will be studied. The aim is to characterize some of the stationary conformational fluctuations that occur in an integral membrane protein, as well as to determine whether the mode changes can be fully accounted for by discrete shifts in the midpoint potential of the activation curves.

The objective of the proposed research is to understand how the structure and function of membrane-spanning channels is determined by their amino acid sequence and by the host bilayer. This question will be examined using the linear gramicidins- a model system that continues to offer a combination of advantages that continues to set it apart from other ion channels: the channel structure is known at atomic resolution, which guides the experimental design and data analysis; one can obtain structural information using single-channel measurements; the channels are permeable only to monovalent cautions, such that single-channel measurements provide direct information about the channels catalytic ability; the channel-forming molecules are made/modified using peptide chemical methods, which allows for convenient introduction of non-genetic amino acids; and gramicidin channels are suitable as molecular force transducers to quantify the energetics of channel/membrane interactions, which provides for a novel way to examine membrane/protein interactions. The experiments address the following questions: what are the molecular determinants of channel folding, membrane insertion, and stability; how does a structural destabilization introduce voltage-dependent gating; how is channel structure and function modulated by the host bilayer; and what are the molecular and structural determinants of the channels' permeability characteristics? These questions will be examined using a combination of single- channel experiments and molecular modeling: single-channel current transitions characterize the channels' catalytic efficiency; the formation of heterodimeric channels, and their relative stability and appearance rates, constitutes a new method to elucidate structural questions pertaining to channel folding, which is especially useful for detecting rare conformers. The experimental results will guide conformational energy and molecular dynamics calculations, which in turn will be used to interpret the experimental results and guide the design of new experiments. The aims are to understand how the amino acid sequence and the membrane environment interact in determining channel structure and function; how a stress introduced by sequence modifications affect the structure and dynamics; how alterations in the host bilayer alter channel function; and how the rate of ion movement, and the ion selectivity, is determined by the channel structure and amino acid sequence.

DESCRIPTION (provided by applicant): Membrane protein function is regulated by the lipid composition of the bilayer in which the proteins are imbedded. This regulation has been described/explained using various terms: changes in bilayer fluidity; changes in bilayer compression or curvature frustration energy, which can be combined into a model of elastic bilayer deformations; changes in lateral pressure profile across the bilayer or in lipid packing stress; changes in bilayer free volume. Among these mechanisms, changes in bilayer can be ruled out, as they cannot explain changes in equilibrium distribution between different protein conformations. The remaining descriptions, though couched in different terms, can be regarded as different approaches to parametrize the lateral interactions among the bilayer-forming lipids and between the lipids and the imbedded proteins. The objective of the proposed research is to examine the energetic consequences of the hydrophobic coupling between membrane-spanning proteins and their host bilayer using an elastic bilayer model, which allows for rather intuitive quantification of the bilayer-protein coupling. In the simplest description, changes in bilayer elastic properties are evaluated in terms of the "pull" the bilayer imposes on an imbedded channel, which can be measured using gramicidin channels as force transducers. In more elaborate descriptions, the coupling can be expressed in terms of an experimentally determined spring constant, which in simple cases can be calculated a priori. The experiments will address the following questions. Can the elastic bilayer model be applied to multi-component lipid bilayers? This will be examined using bilayers composed of two phospholipids with different acyl chain lengths and headgroups, or two phospholipids plus cholesterol, where the spring constant will be determined by changing the hydrophobic mismatch between the bilayer and channel probe. Can the channel-bilayer hydrophobic mismatch "drive" a lateral association between bilayer-spanning channels? This will be studied by examining the relative stabilization of double-barreled channels of different lengths imbedded in bilayer of different thickness. Can pharmacological interventions modulate membrane protein function indirectly, by altering bilayer elastic properties? This will be examined by probing how selected drugs alter bilayer properties, and relating this information to more complex systems. How do changes in bilayer lipid composition alter complex channel function? This will be studied in parallel experiments on gramicidin channels and complex channels reconstituted into planar bilayers of varying composition.

DESCRIPTION (provided by applicant): The Tri-Institutional MD-PhD Program is a joint undertaking of Weill Cornell Medical College (WCMC), The Rockefeller University (RU), and the Sloan-Kettering Institute for Cancer Research (SKI). Its mission is to train biomedical investigators who: on the one hand, have advanced understanding of biomedical science and a mastery of contemporary research skills, which will allow them to undertake fundamental studies to elucidate basic biological processes pertaining to human disease; and, on the other hand, are well grounded in human biology, pathobiology and clinical medicine, which will equip them to transfer advances in basic research to the understanding, prevention, and treatment of human disease. The three institutions operate three graduate schools: Weill Cornell Graduate School of Medical Sciences (WCGS), a joint undertaking between WCMC and SKI; the David Rockefeller Graduate School at RU; and the Gerstner Graduate School of Biomedical Sciences (GSK) at SKI. The Tri-Institutional MD-PhD Program was formed in 1991, when two independent MD-PhD Programs, the WCMC-RU Program and the WCMC-WCGS Program, were joined to form the present program. GSK became part of the Program in 2009. MSTP trainees complete all the requirements for the MD degree at WMC. They will get their PhD training in research laboratories in the three research institutions and receive their PhD degree from GSK, RU or WCGS. Accepted trainees arrive in early July before the start of medical school. During the first two years in the Program they complete their medical school course, take two graduate level courses designed for MD-PhD students, and complete three research rotations (in three different laboratories) before they settle into their thesis laboratory. When te students choose a research laboratory they enroll in the graduate school with which their thesis mentor is associated. The graduate course and thesis requirements are comparable for all MD-PhD students, irrespective of the graduate school in which they are matriculated-and the students can cross register and take courses for credit in any of the graduate schools. At the end of their research training, after they have defended their thesis, the students return to complete their clinical training at WMC. The present application requests funds to continue MD-PhD training at the three institutions beyond Year 40 (the predecessor WCMC-RU Program was formed in 1972; it received NIH funding in 1974). In its current iteration, the Program brings together faculty in more than 270 independent research laboratories. 317 MD- PhDs have graduated from the Tri-Institutional MD-PhD Program and its predecessors; currently there are 108 students in the Program. The current trainees come from 54 undergraduate schools. Their average GPA is 3.75, the combined MCAT score is 35.1. 94% of the graduates pursue post-graduate clinical training; of the 231 graduates who have completed their training, 202 (87%) have appointments in medical schools, research institutes or biotech/pharma.

DESCRIPTION (provided by applicant): The goal of this project is to evaluate computational methods for obtaining in-depth mechanistic insight into ion permeation by tightly combining information from experiments (electrophysiology, thermodynamics and NMR) and computations based on a hierarchical implementation of atomistic models (all-atom molecular dynamics, MD; multi-ion potential of mean force, PMF; and Brownian dynamics, BD). For such investigations to fully exploit the synergism between theory and experiment, the focus must be on a particularly well-defined ion channel. We selected the gramicidin channel because gramicidin channels are small enough that it is feasible to undertake detailed simulations, yet large enough that they have well-defined structural and functional properties - and they are non-trivial in the sense that their permeability properties have proven difficult to simulate quantitatively. Gramicidin channels thus become the system of choice for exploring whether it is possible to develop a comprehensive understanding of ion permeation at the molecular level using atomistic calculations. The proposed studies represent a cooperative effort among three laboratories, which provide complementary expertise to the project and have established records of productive collaborations. The initial computational studies will use a large existing base of measurements of single-channel currents as functions of permeant ion concentration and transmembrane voltage, as a reference for atomistic simulations of channel-mediated ion movement. In parallel, gramicidin analogues will be synthesized to introduce defined perturbations in the PMF for ion movement through the channels: Trp-"Phe substitutions, where the polar Trp is replaced by the nonpolar Phe; Ala-"Ser substitutions, where the non-polar Ala is replaced by the polar Ser. The structural consequences of the substitutions will be determined using solid-state NMR. The functional consequences will be determined using single-channel measurements. These analogues will be subject to MD and BD simulations - of the channel/side chain structure and dynamics, where the NMR results will serve as reference; and of ion permeation, where the electrophysiological results will serve as reference. The transfer free energy of alkali metal cations from water to N-methyl-acetamide will be measured and used to ascertain appropriate representation of ion-peptide group interactions and help calibrate/improve the atomic force field. The proposed research will help establish the range of validity of current models of ion permeation, identify the physical basis of any discrepancy, and implement solutions to resolve any difficulties that are encountered.

The fundamental goal of the Weill Cornell Clinical and Translational Science Center TL1 Training Core Program is to equip pre-doctoral and early post-doctoral trainees, from diverse disciplines, with the skills required to pursue transformative clinical and translational research before their career paths are established. To date, the TL1 Training Program has trained medical and graduate students recruited from all partner institutions. The TL1 trainees are fully integrated into CTSC activities and have access to all services provided by CTSC partners. Building on our successful training program developed over the last 10 years, we will introduce new technologies with an emphasis on team-based research, creativity and entrepreneurship, informatics, and precision medicine. Additionally, we are building more flexibility in training to include an expanded (10 weeks) intensive program for medical students and PhD students, a research experience that will be instrumental in sparking interest and drawing trainees into the world of C/T research. A unique summer program is available for bioengineers to introduce them to clinical research and medicine funded by the Howard Hughes Medical Institute (HHMI). Externships and mini-sabbaticals with biopharmaceutical companies and government agencies will also enrich trainees? exposure to diverse career options in clinical and translational research. To enhance efficiency, pre-doctoral MD and PhD students and pre-doctoral DSN and DPT students at Hunter College School of Nursing will be eligible for dual degree programs during their doctoral training. Flexibility will be built in allowing rolling entry with PACT approval. In this program, trainees can elect an area of concentration in clinical and translational (C/T) research within the Masters Degree in C/T Investigation after completing the core curriculum and a C/T capstone research project. A short-term clinical summer immersion education program will be offered to biomedical engineering PhD students. The overall program will be enriched with workshops, seminars, and courses in creativity, entrepreneurship, team science and informatics. The enhanced TL1 will leverage the trainees to more rapidly advance in the field of Clinical and Translational Science.

PROJECT SUMMARY The Tri-Institutional MD-PhD Program is a joint undertaking of Weill Cornell Medicine (WCM), The Rockefeller University (RU), and the Sloan Kettering Institute for Cancer Research (SKI). Its mission is to train biomedical investigators who: on the one hand, have advanced understanding of biomedical science and a mastery of contemporary research skills, which will allow them to undertake fundamental studies to elucidate basic biological processes pertaining to human disease; and, on the other hand, are well grounded in human biology, pathobiology and clinical medicine, which will equip them to transfer advances in basic research to the understanding, prevention, and treatment of human disease. The three institutions operate three graduate schools: Weill Cornell Graduate School of Medical Sciences (WCGS), a joint undertaking between WCM and SKI; the David Rockefeller Graduate School at RU; and the Gerstner Graduate School of Biomedical Sciences (GSK) at SKI. The Tri-Institutional MD-PhD Program was formed in 1991, when two independent MD-PhD Programs, the WCM-RU Program and the WCM-WCGS Program, were joined to form the present program. GSK became part of the Program in 2009. MSTP trainees complete all requirements for the MD degree at WCM. They receive their PhD training in research laboratories in the three research institutions and receive their PhD degree from GSK, RU or WCGS. Accepted trainees arrive in early July before the start of medical school. During the first two years in the Program they complete their medical school course, take two graduate level courses designed for MD-PhD students, and complete three research rotations (in three different laboratories) before they settle into their thesis laboratory. When the students choose a research laboratory they enroll in the graduate school with which their thesis mentor is associated. The graduate course and thesis requirements are comparable for all MD-PhD students, irrespective of the graduate school in which they are matriculated?and the students can cross register and take courses for credit in any of the graduate schools. At the end of their research training, after they have defended their thesis, the students return to complete their clinical training at WMC. The present application requests funds to continue MD-PhD training at the three institutions beyond Year 45 (the predecessor WCMC-RU Program was formed in 1972; it received NIH funding in 1974). In its current iteration, the Program brings together faculty in more than 250 independent research laboratories. 379 MD- PhDs have graduated from the Tri-Institutional MD-PhD Program and its predecessors; currently there are 137 students in the Program. The current trainees come from 55 undergraduate schools. Their average GPA is 3.75. 94% of the graduates pursue post-graduate clinical training; of the 305 graduates who have completed their training, 258 (85%) have appointments in medical schools, research institutes or biotech/pharma.