Bruce J Nicholson - US grants

The direct metabolic and electrical coupling of cells by means of gap junctions has far significance for the integration of tissue function as a whole and specific relevance to the regulation of cell proliferation. Tumor and transformed cells typically show reduced coupling and many transforming agents (eg. oncogenes, tumor promotors) seem to down-regulate junctional expression. The reverse has also been reported for some anti-neoplastic agents (eg. retinoic acid). Clones (cDNA) to several of the known gap junction proteins have now been isolated and clearly show there to be a family of related proteins showing major differences in their regulatory domains. As a first step, the gap junctional channel structure and locations of functional domains on the protein subunits will be studied and compared between the various subtypes. Initial models will be tested and modified using results from in vitro manipulations (eg. phosphorylation), peptide analyses, and site specific antibodies. Functional effects on the junctions will be monitored electrophysiologically in isolated preparations employing the "tip- patch" technique, or in situ in cultured cells. Refinement of these models will be achieved by expression of the proteins in frog oocytes, and subsequently testing the effects of oligonucleotide directed mutations on intercellular channel function. These studies will serve to better describe channel structure, selectivity (if found in the various subtypes) and modes of regulation. Parallel experiments will use conserved and variable region probes from the junctional cDNA's to determine the approximate size of the gene family and the patterns of expression of the different junction subtypes before and after the transformation process, including their distribution within selected tumors. Finally, the two lines of investigation will be combined by transforming cells with expression vectors containing the junctional cDNA's (in both sense and antisense orientations) and directly testing the necessity or sufficiency of junctional downregulation in the process of transformation and metastatic invasion of issue.

The disruption of intercellular coupling through gap junctions has long been associated with cell transformation and that the promotion phase of tumorigenesis. Transient uncoupling also typically accompanies cases where acute activation of cell growth occurs (e.g. responses to growth factors, tissue regeneration, etc.). Despite this strong of correlation between cell coupling and growth suppression, the mechanism by which this is achieved remains obscure. The problem lies in the broad range of signaling activities that can involve intercellular traffic through gap junctions. Thus, our approach has been reductionistic-first define the properties of different connexins, and their mode of regulation by growth promoting factors such as oncogenes. This in turn will provide the tools with which to investigate the specific mitogenic pathways that are influenced by gap junctional coupling in well defined models of tumorigenesis, such as oncogene induced cell transformation. Given that almost all tissues express multiple connexins, Aim #1 will examine interactions between different connexins, both heterotypic (between cells) and heteromeric (within a cell), how they influence one another and the channel properties that result. We also propose a systematic mapping of exposed residues in the extracellular docking domains of connexins to refine our current model of the structural basis of their heterotypic interactions. Aim #2 seeks to define the gating mechanisms of gap junction channels in response to both voltage and metabolic signals (i.e. phosphorylation). Particular emphasis will be placed on placing the defining mechanism of gap junctional gating by the oncogene v-src. Identification of both the signaling cascades involved, and the specific molecular steps in the gating is proposed. Both of these aims should provide a significantly better understanding of the properties of gap junctions in situ, and should produce tools for Aim #3, in which the molecular mechanisms by which gap junctions suppress cell transformation will be investigated. To provide a define system where the cause of cell transformation is known, and where connexin expression can be controlled for these studies, we have developed an immortalized cell line from Cx43-/- mice. Transformation will be effected by different oncogenes that activate known mitogenic pathways. The efficiency of different connexins in abrogating or preventing this transformation will be assessed, and the specific mitogenic steps that are inhibited by this coupling will be identified. Ultimately, these effects will be correlated with the ability of the different connexins to pass candidate signaling molecules. These strategies are designed to identify both the targets of growth suppressive intercellular signaling, and eventually the signals themselves.

In heart, gap junctions serve the crucial role of electrically coupling muscle fibers to ensure rapid propagation of synchronous contractions. However, the identification of multiple connexins in heart (i.e., Cx37,40,43,45,46) clearly indicates a greater complexity. We and others have demonstrated that intracellular channels comprised of some of these connexins show distinctive gating properties and conductance. Heterotypic interactions between connexins can further modify these properties. This diversity of connexins in heart could then confer subtle (and possibly regional) regulation of electrical conductance or could reflect specializations for additional roles such as metabolic coupling or second messenger transmission. We propose to analyze the nature of the specialized properties conferred by these different connexins and their potential role in cardiac function. Initially, the distribution of these different junctional proteins will be established using both in situ hybridization with highly specific nucleotide probes and antibodies raised to peptides from the deduced sequences in complementary studies on rat and bovine heart sections. Concurrent analyses using the Xenopus oocyte expression system will be aimed at defining the specific properties of intercellular channels comprised of different connexins. Using paired Xenopus oocytes injected with cRNAs of the connexins of interest, two major groups of experiments are proposed. First, the gating properties of the various connexins in response to voltage, pH and Ca++ will be determined using dual cell voltage clamps. Possible heteromeric interactions between connexins and their effects on channel properties will also be investigated. Relating these results to the in vivo distribution of connexins may indicate the manner in which gap junctions modulate the flow of current in the heart. A second series of studies will investigate the passage of larger molecules through junctions. These studies have significance for second messenger responses in heart. In the laboratory of Dr. David Triggle, we will develop a graded series of probes which will allow the determination of exclusion limits and selectivity of junctions comprised of each of the cardiac connexins and their potential hybrid forms. By modifying these probes with respect to surface charge and hydrophobicity, and incorporating photoactivatable cross-linking groups, the nature of the residues lining the channel which may contribute to specificity will also be studied. Site-directed mutagenesis of channel residues will further refine our understanding of the structure of these channels and the molecular basis of any selectivity filters that may be detected. These studies will define the properties of intercellular communication in the heart at a molecular level, so that the roles of junctional proteins in electrical conductance and as regulators of excitability can be addressed.

The broad relevance of gap junctional communication to human health has been graphically illustrated over the last few years by the direct association of no less than 5 very distinct diseases with defects in connexin genes, indicating that the connexin phenotype of a gap junction may impart specialized properties that lead to diverse functions. This contention is strongly supported by the even greater variety of phenotypes that result from genetic ablation of connexins in mice. Although the variability in connexin sequences in the cytoplasmic domains has led to significant interest in their differential regulation, the major function that the gap junctions subserve is the diffusion of low MW metabolites between cells. Specificity in the filtering of these signals by different connexins would certainly impose a whole new layer of complexity to the topic of intercellular communication. This proposal seeks to build on our previous demonstration of selectivity among connexins for ions, larger traces, and natural metabolites by defining the structure of the pore, its gating elements and selectivity filters, and to use this to develop models to explain the mechanism of selectivity. This information is central to understanding the physiological role of gap junctions, and how quite subtle defects in their structure can lead to a diverse array of disease states in man. Specific steps proposed are:(1) Use SCAM, a form of cysteine scanning mutagenesis in which sulfhydryl reagents are introduced and tested for ability to block the channel, to map the residues of connexins that form the pore; (2) Test the accessability of the SCAM channel blocking reagents to different sites in the channel during different gating states to define the physical locations of the channel gate(s) (3) Further characterize the permselectivity properties of connexins with quantitative assays of families of larger probes that vary in specific properties, and, through mutagenesis of the connexin(s), define the sites that confer selecytivity properties for both artificial and natural permeants (4) Develop a 3-dimensional model of diffusion through the gap junction pore using Poisson- Nernst-Planck theory, incorporating structural information on the pore gleaned in Aims 1 and 2.

This application is in response to an RFA for providing Summer Research Experiences in Biomedical Science for undergraduates who come from distinct, non-biological backgrounds. With the increasing need for interdisciplinary approaches to solving biological problems, it is imperative that we modify the way in which the researchers of tomorrow are trained. Particularly, it would be a great asset to attract students with greater quantitative skills. While graduate programs in interdisciplinary areas, such as that run at SUNY at Buffalo by CAMBI, are often available, for many students this occurs too late at a time in which their career track has been set. The current program seeks to intervene during the undergraduate years to expose students, who are currently in more physical science tracks, to the wealth of opportunities provided in the biomedical sciences. An integrated 10 week summer program is proposed that would provide 10 students each year with a research experience in one of several areas, combined with a series of short courses in current topics in the biomedical sciences, a seminar series geared towards career opportunities, and field trips to local research facilities. Research areas represented among the mentors of the program include: tissue bioengineering; mechanisms of pathogenic; molecular analysis of neurological disorders and development; cell biology of inter- and intra- cellular signaling mechanisms; biophysics of ion channels, and; structural analysis of macromolecules. Students in the program would be given an initial orientation to lab techniques, and to the research opportunities available within the program in their first week. At the close of the program, the students will present their work at a meeting of all of the CAMBI labs as part of a research retreat scheduled prior to the beginning of Fall classes. Extensive efforts through a dedicated web page will be used to publicize the results of the student research, and as a means of tracking students who leave the program to assess its effectiveness.

[unreadable] DESCRIPTION (provided by applicant): This application is in response to an RFA for providing Summer Research Experiences in Biomedical Science for undergraduates from distinct, non-biological backgrounds. With the increasing need for interdisciplinary approaches to solving biological problems, it is imperative that we modify the way in which the researchers of tomorrow are trained, particularly incorporating quantitative skills to meet the demands of the new age of bioinformatics, signaling networks, and biophysics. The current program seeks to intervene during the undergraduate years to expose students, who are currently in more physical science, computer and engineering tracks, to the wealth of opportunities provided in the biomedical sciences. An integrated 10 week summer program is proposed that would provide 10 students each year with a research experience entailing a semi-independent project within the lab of their choice (selected from over 40 mentors). The research experience will be enriched through a series of short courses and seminars in current topics in the biomedical sciences (including guest speakers who will present a variety of career opportunities for quantitative biologists), field trips to local research facilities (University centers, biotech, and pharmaceutical companies, etc.), and weekly social interactions among the students. Research areas represented among the mentors range from highly biophysical approaches to structure and function of proteins and DNA, to much more direct medical approaches to research. Students in the program would be given an initial orientation to lab techniques, and to the research opportunities available prior to selection of a mentor in consultation with the steering committee (one student per lab). At the close of the program, the students will present their work at a symposium for students and mentors in the program. Recruiting will occur at a National level through extensive mailings and a user interactive web site. Emphasis will be given to surveying student opinions during the program and tracking their career choices after they leave. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A growing body of evidence, contributed to significantly by work from the previous funding cycle, has demonstrated that gap junction channels display a surprising level of selectivity for their permeants, dictated by the connexin composition of the channels. The basis of this selectivity is complex, but understanding it will be critical to defining the diverse role these intercellular channels play in normal and pathogenic cell function. It is likely that the principles governing selective permeabilities of these channels will be distinct from the much better studied ion channels, and will require a new generation of tools. The current proposal seeks to develop these tools and to apply them to a mapping of the molecular determinants of permeabilty through these channels. A comparative analysis of gap junction channels comprised of different connexins that display very different permeability properties should define the structural diversity within these pores that may underlie their different properties (Aim #1). Scanning and targetted mutagenic and labeling strategies will then be used in the different connexins to map selectivity determinants for size and charge of permeants, as well as mapping sites of affinity for natural permants (Aim #2). We also propose the first comprehensive comparison of peremability characteristics and pore structure of intercellular gap junction channels and their corresponding hemichannels on the cell surface (Aim #3). Initial evidence suggests that there may be significant differences between these two channel isoforms, and growing evidence for the physiological significance of hemichannels in several processes, including cell death via apoptosis, etc., makes understanding such differences critical. The comparative studies proposed here should also resolve a controversy in the literature over the actual nature of the pore lining. While different members of the gap junction family have been mapped as the genetic causes of a surprising variety of human diseases, from catarracts, skin disease and peripheral nerve paralysis to the most common form of hereditary sensorineural deafness, we still have almost no understanding of how damage to these genes can cause the phenotypes observed. The current work will make a significant stride in resolving this issue by defining the molecular structure of these intercellular pores and how this can regulate what goes through them. [unreadable] [unreadable] [unreadable]