Anthony Auerbach - US grants

DESCRIPTION (provided by applicant): The muscle acetylcholine receptor (AChR) is an ion channel that mediates transmission at the nerve-muscle synapse. After binding two transmitter molecules, the AChR switches rapidly (and with high probability) from a closed-channel (C) to an open-channel (O) conformation. With prolonged exposure to agonist, AChRs also adopt inactivated (desensitized) conformations. We seek to understand the dynamics of the molecular events that constitute the binding, gating and desensitization reactions. Results to date suggest that the allosteric gating conformational change is asynchronous, with residues in the extracellular domain of the protein moving in advance of those in the membrane domain during the C-to-O isomerization. Could it be visualized, we hypothesize that this conformational change would appear as a staggering sequence of back and forth motions of a few rigid body domains rather than as a smooth transition between the C and O conformations. Perturbations to the protein (for example mutations that cause the disease slow-channel congenital myasthenic syndromes) alter gating, and, hence, synaptic function, by changing the propagation of this Brownian conformational 'wave'. We will use single-molecule electrophysiology and kinetic (phi-value) analysis to probe the properties of the brief intermediates that constitute the transition state of the gating reaction. Our specific aims are to i) extend the map of phi-values, ii) measure the degree of synchrony between the five AChR subunits, iii) explore the discreteness of the rigid body gating domains, iv) quantify the temperature dependence of the channel-opening speed limit, and v) extend our theoretical analyses of the transition state. We also propose to use similar approaches to study the intermediate states of the transmitter binding and desensitization reactions. The results will illuminate the dynamic machinery of the AChR, and will provide fundamental insight into the mechanisms by which drugs, toxins, cellular perturbations and disease-causing mutations modify ion channel function. They will also serve as the basis for rational protein engineering.

The proposed projects that will benefit from a high performance parallel computer are concerned with the molecular mechanisms of excitability in nerve, muscle, and other cells. Dr. Anthony Auerbach will study the kinetic properties of single ion channel currents elicited by the neurotransmitters acetylcholine or glutamate. Dr. Donald Faber will carry out computer modeling of synaptic currents in conjunction with experimental studies of central glycinergic synapses. Dr. Frederick Sachs will study the structure and dynamics of mechanosensitive ion channels as seen by high resolution image processing of membrane patches. A long term goal of these studies is to understand the molecular processes by which cells regulate ionic fluxes in response to environmental stimuli such as the presence of neurotransmitter molecules of membrane tension. They all require computationally- intense algorithms include matrix operations, Monte Carlo simulations, and three dimensional image processing (deconvolution and tomographic reconstruction). Acquisition of a parallel computer capable of sustaining at least 150 MFLOPS will significantly expedite these calculations.

The NMDA receptor, a subclass of the neurotransmitter glutamate receptors, is widely present at synapses in the vertebrate central nervous system. NMDA receptors have been implicated in the process of long term potentiation, a model of learning and memory, and synapse formation and stabilization. The long term objective of this research is to understand the mechanisms of operation of synaptic ion channels, particulary, the processes of ligand binding and channel gating. The immediate goal of the research is to examine the activation mechanisms of synaptic receptors stimulated by glutamate. By applying kinetic analyses, Dr. Auerbach will be able to investigate how agonists, glutamate- like molecules, activate NMDA ion channels, regulate the gating of channel pores and the desensitization of NMDA receptors. In addition, the developmental changes in NMDA activated channels will be studied. The Principal investigator has developed a method of applying agonist by diffusion through the back of a pipette, a powerful technique which will enable the PI to obtained data from a single channel over a range of concentrations. Experimentally, this approach will allow for the study of ligand activated channels in their native cellular environment. Results of this investigation will contribute to the understanding of the mechanisms of how the glutamate neurotransmitter operates and will provide a microscopic description of synaptic transmission.

9403225 Auerbach This Small Grant for Exploratory Research will conduct a quantitative analysis of ion channel kinetics using higher order hidden markov models (HMM). Ion channels are proteins that regulate the electrical behavior of cells. The investigators will use the HMM method to study these channels, and will simulate these channels on a network of workstations, using parallel codes. This high risk project should help determine the applicability of the use of HMM for understanding ion channel kinetics. ***

The long term goal of this research is to understand the mechanisms by which synapses in the brain are changed by activity and environmental stimuli. We focus on a specific type of synaptic receptor - the NMDA class of glutamate receptor - that plays an important role in long-term changes in synaptic function, the regulation of cell-death, and the generation of patterned activity in neuronal networks. These processes require that the NMDA receptors allow the appropriate amount of calcium ion to pass through its pore and enter the neuron, and be blocked by extracellular magnesium ion over an appropriate range of membrane potentials and concentrations. Thus, in order to understand the molecular bases of synaptic plasticity, we must understand the mechanisms that determine the divalent cation permeability and block of NMDA receptors. We will use recombinant DNA technology to make point mutations in the receptor protein, and then study the altered functions of mutant receptors (expressed in oocytes) using single-channel patch clamp methods. We will investigate how the pore residues, and the subunit composition of the receptor, determine the affinity for calcium and magnesium, if the channel conductance is regulated by electrostatic mechanisms, and the mechanisms by which subconductance levels are generated. We will also investigate the subunit composition (type and copy number) of receptors by studying the patterns of subconductance states that are apparent in receptors composed of different mixtures of wild type and mutant subunits. An understanding of the molecular mechanisms of operation of NMDA receptors channels will provide insight into the basis of developmental, degenerative and other diseases of the nervous system.

DESCRIPTION (provided by applicant): A fundamental principle of biomedicine is that small molecules can bind to specific receptors to trigger physiological responses. We seek to understand the energetic nature of the molecular events that occur at the neurotransmitter binding sites of the neuromuscular acetylcholine receptor when this channel 'gates'between non-conducting and ion-conducting conformations. This knowledge will help us understand the biophysical mechanisms of ligand-protein complexes, and will advance our ability to design new drugs for nicotinic (and other) receptors. Structure-based drug discovery should incorporate the fact that drug action depends on the differential binding of ligands to inactive vs. active conformations of a receptor. To address this point we will use single-channel electrophysiology and kinetic analysis to study unliganded gating, which will allow us to measure all of the salient activation equilibrium constants and to ascertain the energetic contributions of the side chains at each of the two transmitter binding sites. We will also estimate differential binding energies for small ligand probes, in both wild type and mutant receptors. Eventually, this knowledge will be used to engineer acetylcholine receptors that respond predictably to arbitrary ligands. PUBLIC HEALTH RELEVANCE: The experiments outlined in this project establish the fundamental principles for engineering a protein to respond in predictable ways to specific drugs. These principles can be applied to the development of new pharmaceuticals, and to understanding the mechanism by which a drug causes a protein to change shape.

Nicotinic acetylcholine receptors (AChRs) at the nerve-muscle synapse enter and recover from D(esensitized) states that remain inactive in the continuous presence of neurotransmitter. Receptor desensitization down-regulates cell responses and encodes long-term activity patterns. Our goal is to understand the AChR desensitization process at a molecular level. The essential problem of how D states are connected to C(losed) and O(pen) gating states remains unsolved. We will investigate two hypotheses: i) D, O and C are connected in a closed cycle and ii) D is not connected either to C or O but rather to a short-lived gating intermediate state. We will test the second hypothesis experimentally by using mutations and voltage to shift the point of bifurcation between gating and desensitization conformational-change pathways, towards C to produce openings upon recovery or towards O to prevent openings. Many amino acids change structure during the AChR gating and desensitization transitions, and each of these microscopic rearrangements has an associated and mostly-local free energy change. We will measure these energy changes in desensitization at two regions in the AChR transmembrane domain: i) a proline kink in M1 and ii) the hydrophobic M2 gate. Despite intensive investigation, the molecular basis of AChR desensitization has remained obscure since the process was first described 60 years ago. The experiments in this proposal will fill this substantial gap in our understanding of receptor operation.