Designing an Integrated Nanoscale System for Ion Channel Structure-Function Study

Summary

Principal Investigator: Ratneshwar Lal
Affiliation: University of California
Country: USA
Abstract: DESCRIPTION (provided by applicant): Overall goal of this application is to design and develop nanotechnology to study key nanoscale biostructures - Ion channels and receptors that are essential for all living functions. Changes in their three-dimensional (3D) structure and activity, in response to stimuli related to life style, including drug addiction, trigger severe health abnormalities. Understanding 3D structure-activity relationship of these nano-biostructures has been a central and yet elusive goal. 3D structure is currently examined with time and resource limiting X-ray diffraction and EM. Ion channel activity is analyzed by patch clamping and fluorescence microscopy. However, there is no integrated system for a direct 3D structure-activity study of these nano-biostructures in aqueous buffer. Atomic force microscopy (AFM) provides high resolution structural information, in aqueous medium, for many macromolecular complexes, including channels and receptors. AFM is ideally suited to image the surface topology - the primary structural domain where external stimuli, including drug molecules would normally interact. Open architecture of AFM permits integration of other techniques. An integrated multimodal AFM would allow real-time imaging of channel (or receptor)-stimuli (or perturbants) complex, their physicochemical properties and resulting channel conformations. We propose to design a state-of-the-art double chamber AFM integrated with high resolution imaging and permeability assay tools. As a test of its applications related to NIDA's mission, a potential supporter of this application, we will study two important ion channels: hemichannels and Acetyl choline receptor (AChR) that are intimately related to drug addiction. Their 3D structure and their permeability to ions and signaling molecules, in response to drug addiction-inducing stimuli will be examined. Hemichannels connect a cell to its extracellular milieu or its neighbor cells. They are linked to smoking-induced cell pathology and their presence is modulated by drug addiction-related cell receptors (e.g., dopamine receptor) and stimuli. Specific Aims of the application are: 1. Design a combined AFM, Support silicon Chip with a nanopore, TIRF, Single molecule FRET and voltage-sensitive dye imaging systems. As a test of this system, image 3D structure of hemichannels and AChR. 2a. Examine molecular permeability and ionic conductance, in response to physiological and drug addiction-related stimuli. This includes, a) measuring channel permeability to ions, sensor dyes and signaling molecules and b) examining role of defined gating agents and drug addiction-related perturbations, including smoking condensate, nicotine and ROS on the channel permeability, and 2b. Examine density, distribution and turnover of hemichannels and AChR (a drug addiction related receptor) in single cell plasma membrane in response to drugs (e.g., cocaine, nicotine) and pathological agents. The integrated imaging system developed in this study will be first of its kind and will have far reaching and broader role in defining our understanding of the molecular determinants of drug addiction, their pathological consequences as well as in development of therapeutics for drug addiction and treatment. PUBLIC HEALTH RELEVANCE: Consequences of drug addictions on human health and society at large are considerable, yet there is a limited understanding of the underlying mechanism(s) of the cause and/or deleterious effects of these addictions. Most of the drug addiction stimuli possibly induce their effects through them modulating the structure and activity of ion channels and receptors such as acetyl choline receptor (AChR) and gap junctional hemichannels. Currently there is no experimental tool to measure simultaneously an ion channel activity while imaging its 3D molecular structure, the 3D conformations;yet this is the kind of information that is essential to advance our understanding of the molecular mechanism underlying drug addiction and/or their pathological consequences. Advances in nanoscience and technology provide perhaps, the best avenue to explore complex pathological processes that are mediated by nanoscale biostructures, such as ion channels and receptors. Here we intend to implement the most advanced integrated multimodal tools and test their applications on two major classes of ion channels, hemichannels and AChR. Our successful completion of the proposed undertaking will be like combining EM with patch clamping and single molecule imaging that fill the void as well as will provide viable avenues for development of therapeutics for drug addiction and treatment.
Funding Period: 2008-07-15 - 2013-04-30
more information: NIH RePORT

Top Publications

  1. pmc Insulated conducting cantilevered nanotips and two-chamber recording system for high resolution ion sensing AFM
    Brian Meckes
    Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
    Sci Rep 4:4454. 2014
  2. ncbi Graphene nanopore support system for simultaneous high-resolution AFM imaging and conductance measurements
    Laura S Connelly
    Materials Science and Engineering Program, Department of Bioengineering, and Department of Mechanical and Aerospace Engineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
    ACS Appl Mater Interfaces 6:5290-6. 2014
  3. doi An on-demand four-way junction DNAzyme nanoswitch driven by inosine-based partial strand displacement
    Alexander H Mo
    Materials Science and Engineering Program, University of California, 9500 Gilman Drive, La Jolla, San Diego, CA 92093, USA
    Nanoscale 6:1462-6. 2014
  4. pmc High performance, LED powered, waveguide based total internal reflection microscopy
    Srinivasan Ramachandran
    University of California San Diego, Department of Mechanical and Aerospace Engineering, La Jolla, CA 92093, USA
    Sci Rep 3:2133. 2013
  5. pmc Heterogeneous elastic response of human lung microvascular endothelial cells to barrier modulating stimuli
    Fernando Teran Arce
    Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, California, USA Bioengineering Departments, University of California San Diego, La Jolla, California, USA Electronic address
    Nanomedicine 9:875-84. 2013
  6. ncbi Scope of atomic force microscopy in the advancement of nanomedicine
    Srinivasan Ramachandran
    Department of Bioengineering, University of California at San Diego, La Jolla, CA, 92093 0412, USA
    Indian J Exp Biol 48:1020-36. 2010
  7. doi DNA zipper-based tweezers
    Preston B Landon
    Department of Bioengineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
    Langmuir 28:534-40. 2012
  8. doi Potential role of atomic force microscopy in systems biology
    Srinivasan Ramachandran
    Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
    Wiley Interdiscip Rev Syst Biol Med 3:702-16. 2011
  9. pmc Atomic force microscopy of Connexin40 gap junction hemichannels reveals calcium-dependent three-dimensional molecular topography and open-closed conformations of both the extracellular and cytoplasmic faces
    Michael J Allen
    Section of Pulmonary Critical Care, Center for Nanomedicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA
    J Biol Chem 286:22139-46. 2011
  10. pmc Multidimensional atomic force microscopy: a versatile novel technology for nanopharmacology research
    Ratnesh Lal
    Department of Bioengineering, University of California, San Diego, La Jolla, 92093 0412, USA
    AAPS J 12:716-28. 2010

Scientific Experts

  • Ratneshwar Lal
  • Srinivasan Ramachandran
  • Brian Meckes
  • Laura S Connelly
  • Fernando Teran Arce
  • Preston B Landon
  • Alexander H Mo
  • Michael J Allen
  • Meni Wanunu
  • Gennadi V Glinsky
  • Alan L Gillman
  • Max M Yang
  • Joseph Larkin
  • Steven M Dudek
  • Joe G N Garcia
  • Sara M Camp
  • Dosuk Yoon
  • Timothy Gidron
  • Alan Gillman
  • Joanna Gemel
  • Eric C Beyer

Detail Information

Publications10

  1. pmc Insulated conducting cantilevered nanotips and two-chamber recording system for high resolution ion sensing AFM
    Brian Meckes
    Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
    Sci Rep 4:4454. 2014
    ..These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels. ..
  2. ncbi Graphene nanopore support system for simultaneous high-resolution AFM imaging and conductance measurements
    Laura S Connelly
    Materials Science and Engineering Program, Department of Bioengineering, and Department of Mechanical and Aerospace Engineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
    ACS Appl Mater Interfaces 6:5290-6. 2014
    ..The functionality of this integrated system is demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore and simultaneous AFM imaging of the bilayer. ..
  3. doi An on-demand four-way junction DNAzyme nanoswitch driven by inosine-based partial strand displacement
    Alexander H Mo
    Materials Science and Engineering Program, University of California, 9500 Gilman Drive, La Jolla, San Diego, CA 92093, USA
    Nanoscale 6:1462-6. 2014
    ..This approach can provide structural organization and spatially control other multicomponent molecular complexes. ..
  4. pmc High performance, LED powered, waveguide based total internal reflection microscopy
    Srinivasan Ramachandran
    University of California San Diego, Department of Mechanical and Aerospace Engineering, La Jolla, CA 92093, USA
    Sci Rep 3:2133. 2013
    ..The simple design allows integration with other imaging systems, including atomic force microscopy (AFM), for probing complex biological systems at their native nanoscale regimes. ..
  5. pmc Heterogeneous elastic response of human lung microvascular endothelial cells to barrier modulating stimuli
    Fernando Teran Arce
    Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, California, USA Bioengineering Departments, University of California San Diego, La Jolla, California, USA Electronic address
    Nanomedicine 9:875-84. 2013
    ..Our finite element analysis results substantiate this approach. The heterogeneous elastic behavior correlates with differential cytoskeletal rearrangements observed with fluorescence microscopy...
  6. ncbi Scope of atomic force microscopy in the advancement of nanomedicine
    Srinivasan Ramachandran
    Department of Bioengineering, University of California at San Diego, La Jolla, CA, 92093 0412, USA
    Indian J Exp Biol 48:1020-36. 2010
    ..In this context, AFM and its variants play a pivotal role in contributing towards the nanomedicine knowledge-base that is required for fruitful developments in nano-diagnostics and nano-therapeutics...
  7. doi DNA zipper-based tweezers
    Preston B Landon
    Department of Bioengineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
    Langmuir 28:534-40. 2012
    ..Our approach yields a robust, compact, and regenerative tweezer system that could potentially be integrated into complex nanomachines...
  8. doi Potential role of atomic force microscopy in systems biology
    Srinivasan Ramachandran
    Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
    Wiley Interdiscip Rev Syst Biol Med 3:702-16. 2011
    ..Atomic force microscopy (AFM), AFM cantilever sensors, and AFM force spectroscopy in particular, could address these issues directly. In this article, we reviewed and assessed their potential role in systems biology...
  9. pmc Atomic force microscopy of Connexin40 gap junction hemichannels reveals calcium-dependent three-dimensional molecular topography and open-closed conformations of both the extracellular and cytoplasmic faces
    Michael J Allen
    Section of Pulmonary Critical Care, Center for Nanomedicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA
    J Biol Chem 286:22139-46. 2011
    ....
  10. pmc Multidimensional atomic force microscopy: a versatile novel technology for nanopharmacology research
    Ratnesh Lal
    Department of Bioengineering, University of California, San Diego, La Jolla, 92093 0412, USA
    AAPS J 12:716-28. 2010
    ..Finally, we will discuss some future directions including AFM tip-based parallel sensors integrated with other high-throughput technologies which could be a powerful platform for drug discovery...