Francisco Bezanilla - US grants

The object of this project is to describe and characterize the molecular basis of voltage dependent processes. In particular, the aim is an understanding of the physical events responsible for the voltage dependence of sodium and potassium channels and the sodium/calcium exchange. The experiments use electrophysiological techniques to describe the currents through a large number of channels (macroscopic currents), through a few channels (noise measurements), through single channels and the currents produced by the rearrangement of the charged groups of the channel macromolecule. Channels will be modified (BTX for sodium and ATP for potassium) in an attempt to gain more information about their physical states. Results will be interpreted using kinetic models with energy barriers between the physical states represented by the positions of the charged particles in the macromolecule. Parameters will be fitted to these models using all the information from the electrical measurements and they will be modified according to the results of the fitting. In addition, labelled ATP will be used to mark the K channel and attempt its extraction and characterization. The Na/Ca exchange will be characterized by measuring isotopic fluxes under membrane potential control and the currents produced by the exchange. We will attempt the determination of its stoichiometry and voltage dependence. Experiments will be performed using the giant axon of the squid in perfused, dialyzed or cut-open configurations. This latter technique allows patch clamping from the internal side of the membrane. The experiments requiring larger amounts of membrane material will be performed in membranes extracted from the retinal nerve of the squid. This research is expected to provide new information on the detailed mechanisms of voltage gated channels and on the operation of the sodium/calcium exchange which will help in the understanding of the molecular mechanisms of voltage dependent processes. These studies are expected to have relevance in the physiology of nerve, muscle, excitable tissues and cells in general.

[unreadable] DESCRIPTION (provided by applicant): The long term objective of this project is the development of an optical detection system based on surface plasmon resonance to study the dynamics of membrane proteins with special emphasis in voltage gated ion channels such as the Na and K channels that are responsible for the generation and propagation of the nerve impulse. Channel proteins are labeled in specific sites with fluorescent probes using cysteine chemistry and the fluorescence is detected by the proposed optical setup. Fluorescence changes, produced by quenching or energy transfer are indicators of local environmental changes and thus they follow conformational changes within the protein as the channel undergoes transitions from the closed to the open state. The optical apparatus uses a hemispherical lens that couples an incoming laser beam on a glass chip that has a thin (50 nm) siver layer where the biological preparation lies separated by a thin (10 nm) layer of silicon oxide. The correct angle of excitation induces plasmon resonance in the metal and enhances the fluorescence of fluorophores labeling the channel. The detection is done from the biological preparation side or from the excitation side. In the second case the signal to noise ratio is expected to be much larger because the coupled emission comes from a region limited to 20 nm and, as it is directional, a specially designed optics collects most of the light on a photodetector. The testing of the optical sytem is done on labeled ion channels expressed in mammalian cells or in supported bilayers. In the second case, the supported bilayer is made with liposomes containing purified labeled channels. The voltage across the bilayer is changed taking advantage of the silver layer of the plasmon chip. A modification of the optical system is also proposed to image the biological preparation to follow the time course of the fluorescence of individual molecules in response to voltage pulses that change the conformation of the channel. The understanding of conformational dynamics of channel proteins is a crucial step in the design of drugs or therapies needed to ameliorate or cure several neurological deseases produced by abnormal function of ion channels. The optical system developed in this application is aimed at developing a new microscope that is especially designed to detect conformational changes of ion channels with improved resolution, higher sensitivity and improved rejection of spurious fluorescence than presently available devices. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The sodium potassium (Na+/K+) pump is an important membrane protein that uses the energy of ATP to transport Na+ and K+ against their electrochemical gradients to maintain cell homeostasis. This study is aimed at the molecular understanding of the ionic events in the Na+/K+ pump transport cycle. Specifically, we aim at the description and quantification of the movements of Na+ and K+ in and out from the solution to the binding sites located within the pump protein. The experiments consist of measuring the charge movement that results from voltage perturbations using a fast voltage clamp in the squid giant axon. These experiments allow the measurement of the time course of the movement of each of the three Na+ ions released from the pump into the external solution. This proposal is to be carried out at the Laboratory of Cellular Physiology in Montemar, Chile, where 1 mm axons from the squid Dosidicus gigas are available. With these axons, the signal-to-noise ratio of the charge movement is much higher than in the Loligo pealeii axons, which will allow measurements that are not feasible with Loligo axons. The specific aims are 1) understand the thermodynamics of extracellular Na+ translocation transitions. We aim at the study of the entropic and enthalpic changes that occur during the binding and release of the Na+ ions in and out of the external access channel of the pump. This will be done by measuring the temperature dependence of all three components of the charge movement associated with the Na+ translocation. 2) Understand the biophysical properties of K+ translocation mechanism of the Na+/K+ pump. This specific aim requires a comprehensive study of transitions involving the movement of K+ as a function of external concentration of K+, voltage, intracellular ATP and inorganic phospate. These studies are expected to give a mechanistic and molecular description of the electrical events mediated by the Na+/K+ pump, which is a ubiquitous membrane protein of fundamental importance in health and disease. This research will be done primarily in Montemar, Chile in collaboration with Ramon Latorre, Professor, Universidad de Valparaiso, as an extension of NIH grant No R37GM30376-29. PUBLIC HEALTH RELEVANCE: The experiments proposed here are expected to give direct measurements of the ion movements in and out of the Na+/K+ pump and provide the biophysical mechanisms of the ion binding and unbinding during the pump operation. The activity of the Na+/K+ pump is vital for cell homeostasis because it helps maintaining the Na+ and K+ gradients. It is especially important in excitable cells where the loads change during the action potential. Clinically, the Na+/K+ pump is important because it is the receptor of digoxin, digitoxin and other cardiotonic steroids.

The long term objective of this proposal is the understanding of voltage sensing in membrane proteins at the molecular level. Voltage sensing plays a major role in excitable tissues such as nerve, muscle and heart. We propose to study how voltage changes induce conformational changes in voltage gated sodium and potassium channels and in the voltage sensitive phosphatase Ci-VSP by correlating the gating currents with simultaneous rearrangements followed by fluorescence changes of probes placed in specific sites. Ultimately we would like to reproduce the function of the proteins with the landscape of energy obtained from structural changes. In this period we have three specific aims. Aim 1: Correlation of voltage sensor regions with the energy landscape of activation. Using Shaker K channels as a model we will study the trajectory of the gating charges through the hydrophobic plug using a fluorescent replacement of arginine that is quenched by Trp. Site-directed electrochromic fluorometry will be used to test the local field in different states of the sensor during gating. The interactions of the charges with the plug will be studied during activation and deactivation as a function of time and voltage. We will look for possible movements of S2 and S3, try to define the differences between the activated and relaxed states using LRET, and search for conditions that stabilize the relaxed state. Aim 2: Correlation of conformations and kinetics with Ci-VSP structures. Recent crystal structures of Ci-VSP in putative resting and activated/relaxed states will be compared and correlated with the function and structural changes detected during transitions to address how many charges move per molecule, the size of the individual shot of charge during gating, the effect of the residues of the hydrophobic plug on kinetics and steady-state, a possible secondary structure change of S4 during gating, and recording of single sensor movements by single molecule fluorescence of purified and reconstituted proteins or expressed in oocytes. With aims 1 and 2 we expect to obtain general rules of conformational changes but also specific differences between Shaker and Ci-VSP. Aim 3: Conformational changes and kinetics of the eukaryotic sodium channels. In this period we aim at two general objectives. First, by using LRET with new fluorescent toxins and site- directed fluorescence we will measure conformational changes of each individual domain of Nav1.4 as a function of voltage with and without the ?1 subunit. Second, we will define the molecular basis of the fast kinetics of Na channels induced by the beta subunit, which is crucial for action potential generation, We will test the hypothesis that the ?1 subunit induces positive cooperativity. We will measure the distances between the ?1 subunit and ? subunit with LRET, and determine the number of ?1's per ? with LRET and single molecule fluorescence. This research is expected to impact our knowledge of how specific residues affect kinetics and steady-state properties that in many cases can be traced to mutations that cause epilepsy, arrhythmia, myotonias and sudden death.

P-class (or E1-E2-type) ATPases constitute a superfamily of cation transport enzymes, present both in prokaryote and eukaryote, whose members mediate membrane flux of all common biologically relevant cations [1]. P-class pumps use ATP to transport ions against a gradient. The sarcoplasmic reticulum Ca+ -ATPase (SERCA) pumps 2 Ca+ from the cytosol of muscle cells to the sarcoplasmic reticulum by exchanging H"". In each normal cycle, the Na/K pump transports 3 Na" out of the cell by 2 K = into the cell at the expense of the hydrolysis of one molecule of ATP. This extended bridge project about P-class ATPases represents the combination of two bridge projects, focused on the Na/K and SERCA pumps. From crystallographic snapshots of SERCA and the recently solved first crystal structure of the Na/K pump, we now dispose of a remarkable series of pictures showing how these enzymes look at different states of their transport cycle. SERCA is now by far the membrane protein where the most functionally different conformations have been described in precise structural detail [4, 5]. However, electrophysiological studies with SERCA are harder to do than with the Na/K pump, which can be expressed heterologously on the surface of cells. For this reason, while structural information about the Na/K pump is more limited (with only one x-ray structure available), the most informative functional analysis comes from experiments on the Na/K pump. In this proposal, we address questions about the Na/K and SERCA pumps: What are the conformational dynamics involved as the pump transits through conformational states revealed by x-ray crystallography? What is the nature of the coupling between the binding of ATP, phosphorylation, and the movements of charged species across the core of the protein? What are the stepwise voltage-sensitive steps? What is the complete reaction pathway and what are the individual transition rates under various physiological conditions? Answers to these questions will correlate the structural changes to the function of membrane P-class ATPase pumps.

DESCRIPTION (provided by applicant): Fluorescence spectroscopy has made myriad essential contributions to our understanding of molecular and cellular biology. Fundamental properties of intrinsic and extrinsic fluorophores probes in biological systems are exquisitely sensitive to the local bimolecular environment on (sub)-nanometer length scales. Measurement of fluorophore probe properties such as excited state fluorescence lifetime decay, polarization anisotropy, and energy transfer can reflect detailed structural, conformational, motional, binding, and dynamical interactions of macromolecules such as proteins, enzymes, RNA, and DNA. For instance, small changes in the fluorescence lifetime of a probe reveal the characteristics of processes in the local environment at short distances under 10 E (e.g., quenching and dielectric contributions) and long distances up to 100 E (e.g., Forster resonance energy transfer or FRET). This allows direct determination of important parameters of protein-protein binding, complex formation, molecular contact, proximity and overall conformational changes. These lifetime measurements require a highly sensitive lifetime fluorometer with picosecond resolution, such as the requested ISS ChronosBH Fluorescence Lifetime Spectrometer. This automated instrument combines the robust, fast, and proven Time-Correlated Single Photon Counting (TCSPC) method with a novel white light continuously tunable picosecond pulse excitation source that covers the entire visible to near-IR spectrum. The instrument can quickly measure multi-component lifetimes from 40ps to 1us (and longer in phosphorescence mode) with high fidelity from small volume (30uL) and weakly fluorescent samples. Kinetics studies are possible with a stop-flow apparatus. The instrument enables innovative new research as exemplified by 11 different projects from 9 major users. These projects encompass areas as diverse as molecular motions in ion channels, conformational changes in recombinase DNA complexes, amyloid proteins, catalysis regulation of human insulin degrading enzyme, and chemical synthesis of proteins for the development of new fluorescent probes. The need is urgent as there is no instrument of this capability at the University of Chicago or even the greater Chicago area. To increase availability and impact, the instrument will be placed in the interdisciplinary NanoBiology Facility shared core in the Institute for Biophysical Dynamics. Furthermore, it will be integrated in Graduate Biophysics lab curricula, thereby maximizing its exposure and impact on future biological and biomedical researchers.

The objective of the Instrumentation Spectroscopy Core (SIC) D2 is two-fold: first, detailed in D2.2 Planned Direction of Development, is to improve presently available instruments, develop new instruments, or methods and procedures aimed at improving on signal to noise ratio, specificity and time resolution of spectroscopic techniques and combining them with functional techniques, such as electrophysiology. The second objective, detailed in D2.4 Component to the MPSDC, is to provide service to the MPSD members, and the community at large, with the new developments as well as with the spectroscopic and functional techniques presently installed. Two major areas of development will be pursued. Electron paramagnetic resonance (EPR) and fluorescence provide complementary information on the dynamics of structural changes in membrane proteins. Time-resolved EPR techniques, such as rapid freeze quench (RFQ) will be a priority, together with the development of a microfluidic-based RFQ apparatus. Likewise, we plan to take advantage of the large range of time scales probed by fluorescence. We will do so by optimizing the probes and perfecting the detection techniques (both ensemble and single-molecule) that enable the tracking of dynamic processes in membrane proteins. While the finite photon flux and photostability of single-fluorophores typically limits single-molecule imaging techniques to the ms regime, we will push this boundary to the µs regime through the development of intramolecularly stabilized organic fluorophores. These general goals will be carried out through a series of specific projects: AIM 1: To further develop and perfect a microfluidic rapid freeze quench (?RFQ) EPR system to enable measurements of frozen samples that are generated by rapid mixing of reactants and make ?RFQ accessible to members of the consortium. This technique will be applied in conjunction with double electron-electron resonance (DEER) and Electron nuclear double resonance (ENDOR) experiments. AIM 2: To further develop and enhance single-molecule fluorescence techniques: a) Test, expand and make available high-performance organic fluorophores. Test those that are developed with unnatural amino acid technologies in core D1. b) Establish a setup that combines magnetic tweezers with single-molecule fluorescence. This technique will be used to apply force to membrane proteins to study conformational changes on individual molecules while assessing their functionalities with fluorescent probes. c) Develop a multi-color single-molecule FRET setup that will allow detection of synchronized or correlated motions among multiple domains. d) Develop a setup to measure single-molecule fluorescence with enhanced time resolution to resolve fast conformational changes AIM 3: To further develop and enhance ensemble fluorescence techniques: a) Improvement of an LRET setup and make it available to members of the consortium to measure distances in functional membrane proteins. b) Develop a setup to measure nanosecond fluorophore lifetimes in the microsecond time scale combined with electrophysiology. c) Improve the fluorescence detection system with a new design of the photodetector-to-voltage transducer and develop a new more powerful acquisition system that will be used for all of the above setups.

ABSTRACT Retinal ganglion cells (RGCs) convey visual signals to the brain in the form of action potentials, the initiation and axonal propagation of which depend on the activation of RGC voltage-gated sodium channels (NaVs). In photoreceptor degenerative diseases such as age-related macular degeneration (AMD), inner retinal neurons including RGCs in many cases remain intact and capable of generating action potential responses. RGCs thus represent a logical target for approaches aimed at restoring vision in advanced-stage AMD and related retinal diseases, by bypassing the nonfunctioning rod and cone photoreceptors and establishing direct RGC responsiveness to light. In a recent study of dorsal root ganglion cells (a non-retinal neuron widely studied as a model action-potential-generating cell type) and in hippocampal slice preparations, we have shown that gold nanoparticles (AuNPs) conjugated with a cell-targeting biomolecule enable robust light-induced NaV activation and resulting action potential generation. Essential features of the cell-targeted AuNP technique are: (i) functionalization of the light-absorbing AuNPs to localize them at or near the NaVs; (ii) upon the AuNP?s plasmon absorption of a millisecond/submillisecond light flash, AuNP radiation of the light energy as a nondamaging pulse of heat that creates a localized, transient, depolarizing capacitive current across the plasma membrane; and (iii) resulting activation, i.e., channel opening, of neighboring NaVs and thus action potential initiation by this depolarization. In this application we propose exploratory research to apply this AuNP approach to RGCs in the living eye of the rat. The project?s goal is to establish AuNP treatment conditions that achieve robust AuNP-mediated RGC photo-responsiveness in vivo. In rats for which rod and cone photoreceptor signaling to RGCs has been suppressed pharmacologically, we will intra-vitreally deliver AuNP conjugates designed for binding to the immediate vicinity of the RGC NaVs. Following treatment with the AuNP conjugates, we will employ in vivo recording of electroretinographic (ERG) signals associated with RGC activity, and of visual evoked potentials (VEPs), to analyze properties of RGC electrophysiological responses to AuNP photo-excitation. The research will involve variation of the size/structure of the AuNP, of the AuNP- conjugated RGC-targeting component, and of the duration and energy [(intensity) x (duration)] of AuNP- excitatory flashes. Accompanying the in vivo experiments will be all-optical stimulation/recording of AuNP- mediated action potentials in isolated rat retina, and histological analysis of retinas treated in vivo and in vitro with AuNPs. Results of the in vitro retina experiments will guide the selection of in vivo treatment conditions to be systematically investigated and facilitate interpretation of the in vivo data. Leading the research will be David R. Pepperberg, PhD (Univ. of Illinois at Chicago) and Francisco Bezanilla, PhD (Univ. of Chicago).