Mauricio Montal - US grants

Our ultimate goal is to understand the molecular basis of the process of visual phototransduction performed by visual pigments. Our approach is to solubilize and purify rhodopsin from both vertebrate and invertebrate and then to reassemble it into model lipid bilayers that allow measurements that are not readily achieved otherwise. Rhodopsin has been incorporated into planar bilayers which separate two aqueous compartments and are readily amenable for electrical measurements. Furthermore, asymmetric bilayers containing rhodopsin only on one monolayer are formed by apposing a lipid monlayer to a rhodopsin-lipid monolayer. The effect of light on this system is to induce the formation of a voltage-sensitive channel. The kinetics of channel opening and closing, and the ion-selectivity of the channel are under current investigation. Rhodopsin is also incorporated into bilayer vesicles, with diameters of several micrometers. The optical spectral properties of rhodopsin in the vesicles are similar to those recorded in retinal rod disc membranes by chemical, optical and electrical techniques.

This study aims to test the hypothesis that the hyperexcitability of neuronal membranes in neuropsychiatric disorders which involve disturbances of movement, mood and cognition arises from the ion-channel activity of an endogenous tetanus toxin-like molecule. The hypothesis formulated is unique in so far as it focuses on the clinical and pathophysiological similarities between some forms of schizophrenic psychosis and epilepsy with tetanus, and its novelty is the distinct suggestion of the existence of an endogenous peptide that mimics the known channel activity of tetanus toxin. The experimental strategy is based on the critical concept that a specific entity of predicted amino acid sequence adopts an amphipathic alpha-helical structure in neuronal membranes and aggregates to provide a polar pathway for ionic conduction through the non-polar interior of the bilayer membrane.

This study aims to address the question of the molecular mechanism for the inhibition of ionic conduction through voltage-dependent and neurotransmitter regulated channels by local anesthetics (LA). Specifically, we aim to probe the nature and the site of the blocking action of LA on voltage-dependent sodium channels and nicotinic cholinergic receptors. Our approach is to characterize the action of a series of LA on be single channel properties of synthetic channel peptides in lipid bilayers. The synthetic channel peptides are designed to mimic the channel lining of the mammalian brain voltage-dependent sodium channel with amino acid sequence -- DPWNWLDFTVITFAYVTEFVDL-- and of the nicotinic cholinetic receptor from fish electric organ with the sequence: EKMSTAISVLLAQAVFLLLTSQR. A series of LA well characterized on voltage-gated sodium channels and end-plate channels will be determined. The effect of LA on channel conductance, ionic selec- tivity and saturation will be evaluated. The modification of channel gating kinetics in terms of the number of channel open and closed states or the lifetime of the channel on each of these states will be assessed. The accessibility of the 2 aqueous compartments separated by the bilayer will allows to examine the pH-dependent action of these drugs under symmetric and asymmetric conditions. The concentration dependence, voltage dependence, temperature and calcium dependence of the channel block will be explored. The stereopotency ratio for block will be examined. The results will be compared with data on authentic channels. A salient advantage of this approach is that, by chemical synthesis, an amino acid thought to be crucial for the action of LA can be substituted. The assay of the "analogue" will establish if such residue is significant. Cases in point are the analogues of the sodium channel peptide in which the acidic residues D7 or E18 (located in the pore lumen), D1 (pore entry) or D21 (pore exit) are substituted or the uncharged amino acids N or Q, respectively. Likewise, for the receptor peptide, the channel activity of analogues in which the polar residues S8 and T5 (located in the pore lumen) are replaced for L and El or K2 (pore entry) are substituted for Q and A, respectively, will be analyzed. This program may lead to identify the molecular structures that determine the pharmacological specificity in the action of LA on channel proteins and will provide information conducive to formulate a common mechanism for the inhibitory action of the clinically important local anesthetics on voltage-dependent channels in neural and cardiac cells.

This is a request for an ADAMHA Research Scientist Award (RSA). The brain is an information processor that generates behavior. The functional units for information transfer are the ionic channels in the neuronal membrane. This study aims to test the hypothesis that an etiological component underlying neuropsychiatric diseases such as affective disorders and schizophrenia may be a dysfunction of channel proteins. Our immediate goal is to understand how a channel protein works. To elucidate channel protein structure-function relationships a multidisciplinary yet focused approach encompassing techniques of membrane biophysics, molecular biology and protein engineering will be implemented. The strategy requires the primary structure of the protein to apply empirical secondary structure predictors in order to postulate a structural model. It is then followed by the design and synthesis of peptides proposed to be transmembrane functional components of the assembly (i.e. the "pore" or the "sensor") and the functional assay of the synthetic channel in lipid bilayers. A salient advantage is that, by chemical synthesis, an amino acid thought to be crucial for function can be substituted. The assay of the "analogue" will establish is such residue is functionally significant. Concurrently, small perturbations in the structure of the protein will be produced by site directed mutagenesis of the gene followed by the functional assay of the mutant channel in membrane patches of Xenopus oocytes. The synthetic and the recombinant strategies complementing each other should provide an even more powerful path to establish structure-function relationships in channel proteins. This program will be initially focused on the voltage sensitive sodium channel and the nicotinic cholinergic receptor as prototypes of two major and distinct gene families in the brain. The ultimate goal is to blend psychiatry with the biology and chemistry of brain channel proteins as focused on structure-function correlations to gain new insights into the etiology of mental disorders. The direction of my professional growth has been and will continue to be guided by the goal of my research for the past 20 years: To reduce the complexity of the brain form and function to the language of chemistry.

The ultimate goal of the program is to identify the fundamental principles that determine the biological design of channel proteins. The immediate goal is to realize the molecular design of a pore-forming structure and to use it towards understanding the molecular basis of ionic selectivity, channel blockade and voltage regulation of channel open probability. The central notion is that given the primary structure of channel proteins it may be possible to identify functional modules that will fold predictably into stable structural motifs and fulfill functional attributes of the authentic system. A first step in this endeavor is to model only the most fundamental unit of function of ion channels, namely the pore-forming structure. A plausible molecular blueprint for the pore-forming structure of channel proteins is a bundle of amphipathic alpha-helices that cluster together to generate a hydrophilic channel. Such pore structures are designed from functional modules that represent the amino acid sequence of authentic proteins and refined to accommodate specific functional characteristics. The next level of complexity incorporates the voltage- sensing device and considers the design of a minimum voltage-gated channel that would exhibit the essential pore properties of ionic selectivity with the additional regulation by transmembrane potential. The approach involves: (1) Formulation of a structural model of the protein based on sequence analysis and secondary structure predictions; (2) Conformational energy calculations to assess the validity of the proposed structural motifs, to design "computer mutations" to guide experimental design, and to obtain quantitative descriptions of the energy profile for ionic diffusion through the designed channels; [3] Synthesis of the designed structures by solid-phase peptide synthesis and by direct expression of synthetic genes encoding the designed channel proteins; [4] Functional analysis of the designed channels by reconstitution of synthetic proteins in planar lipid bilayers and by expression of corresponding cRNA in amphibian oocytes or cDNA in mammalian cells. Single channel current recordings under voltage-clamp conditions provide a detailed set of functional parameters to determine ionic selectivity, pharmacological specificity and voltage-dependent regulation of channel open probability; (5] Site-selective replacements for evaluation of structure-function relationships; [6] Protein structure determination by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated detergent micelles and by solid-state NMR in oriented phospholipid bilayer lamellae. It is anticipated that the convergence of structural information with the detailed analysis of channel protein function at the level of single molecular events, integrated with the benefits of peptide synthesis and recombinant DNA techniques, guided by molecular modeling, will provide paths for a systematic investigation of the structure-function map of channel proteins, and may provide clues about the biological design of this class of proteins that are fundamental components of living cells.

proteins; nervous system; nuclear magnetic resonance spectroscopy; biomedical resource; biological products;

The purpose of this facet of the program is to characterize the channel properties of recombinant human immunodeficiency virus type 1 Vpu protein in order to build up the functional data base required to seek structure-function relationships. Realization of the full power of this approach requires a high resolution structure. Thus, the functional information will be combined with the structural data obtained by NMR spectroscopy, X-ray crystallography, neutron diffraction and conformational energy calculations. The strength of the program is based on the multidisciplinary approach focused on this single molecular entity that appears to be important for virus release from HIV infected cells. Biophysical characterization of the channel properties of Vpu involves reconstitution of the recombinant protein in lipid bilayers. These properties include single channel conductance, ionic selectivity, saturation, and open and closed channel lifetimes. Channel blockade will be used to screen for potential blockers. The significance of specific residues in determining the channel activity of Vpu and its sensitivity to blockers will be evaluated by designing and synthesizing site-specific replacements. The contribution of the glutamic acid 2 at the N-terminal end, and of serine 23 at the C- terminal end of the Vpu transmembrane domain to the cationic selectivity determined for the Vpu transmembrane peptide will be examined by substitution for glutamine or alanine, respectively. Channel formation is envisioned to arise from the oligomerization of Vpu. The residues exposed to the channel lumen of the oligomer will determine the permeation and blockade properties of the membrane- embedded Vpu channel. The structure of Vpu and the model calculations will suggest candidate residues for further mutagenesis followed by functional analysis after reconstitution of the mutant protein in lipid bilayers. This cycle of refinements will provide a bluepring for the pore-forming structure that should be pivotal for the design of Vpu-specific channel blockers. The ion channel activity of Vpu, therefore, provides a potential target for drug intervention in the management of AIDS based on the development of Vpu-specific channel blockers.

Voltage gated channels are one of the ways that materials move into and out of cells. They are important across biology. A channel can either be open or closed; the transition is called gating. However, these proteins do not crystallize easily and two-dimensional methods have had to be developed to consider their structure. Each channel protein consists of six hydrophobic membrane-spanning segments and a membrane insertion region.

The ability to predict or at least have good models for these transmembrane proteins is a significant need for the research community. The techniques for this are now just beginning. At the moment, there are no good homologues to VGC proteins in the PDB, because of the difficulty of crystallization. However, alignment of sequence is possible. Three-dimensional protein homology models will be developed from these aligned sequences, using a variety of software packages developed by the group. The resulting tools will be available on the web. Once the first models are made, all similarly aligned proteins can be automatically predicted. This effort will produce models that could be used to refine the structure data coming from new studies and methods of considering membrane proteins.

DESCRIPTION (provided by applicant): The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. This is precisely the gap in knowledge the program intends to fill. The ultimate goal of this endeavor is the understanding of the mechanism of voltage sensing based on the modular design of voltage-gated channel proteins. Major objectives are: to define the protein fold(s) best suited to fulfill the pivotal function of voltage sensing; to delineate a minimum set of determinants sufficient for sensing; to uncover a molecular blueprint for a versatile voltage sensor design for which a finite number of specified perturbations would adapt it to sense a wide range of membrane potential; and to establish the surface compatibility underlying the interaction between the two modules and the propagation of change from one module to the other that produces the exquisite sensitivity of the pore to voltage in intact voltage-gated channels. We propose to characterize the channel properties of the isolated voltage sensor module (VSM), the pore module (PM), and the self-assembled [VSM-PM] complex by overexpression and reconstitution into lipid bilayers and giant proteoliposomes, aiming to recapitulate the functional features of the intact voltage-gated K+ channel (Kv) from its component modules. We propose to explore the voltage sensor sequence landscape approached by generating and screening random libraries of VSM mutants aiming to identify and demonstrate unsuspected channels with new voltage-gating phenotypes. We intend to determine the atomic resolution-structures of KvLm and its modules by X-ray crystallography. Exciting results have already emerged which pave the way for a decidedly productive phase of the program. Overall, the itinerary entails going from modules to sequence, to structure and back to mechanism. This focused and realistic program outlines a novel way of thinking about voltage sensing. PUBLIC HEALTH RELEVANCE: Ion channels, a special class of membrane proteins that allow the selective and regulated diffusion of ions across membranes, are fundamental for cell function and regulation. Their design is a marvel of protein chemistry and evolution, and their dysfunction is at the root of devastating human diseases such as epilepsy and arrhythmia. The voltage sensor, the unique element that endows Na+ and K+ channels, which underlie the nerve action potential with the exquisite sensitivity to transmembrane voltage, remains enigmatic and needs further study. The thrust of our program aims to establish structure-function relationships with a primary emphasis on the modular design of the transmembrane domain in voltage-gated channel proteins.