Tissue and organ function, in health and disease, is determined by the collective activity of cellular processes at the macroscale. Cellular activity underlying organ and tissue function is defined by the nanoscale distributive and cooperative behavior of groups of ion channels that respond to a diverse range of chemical and electrical signals. Ion channel behavior at high resolution remains poorly understood and there are limited techniques for studying the distributed and cooperative properties of ion channel ensembles with single channel resolution, in a physiological environment.
The aim of this research project is to design a novel scanning ion conductance microscope (SICM) probe capable of molecular resolution imaging of the conductance and structure of multiple points simultaneously. This is accomplished through the creation of a cantilevered SICM-array featuring sharp conducting tips with low spring constants, which allow for imaging of soft biological membranes. To get localized electrical conduction images, only the metallic tip apex will be conducting with the remainder of the cantilever completely insulated. Each conducting cantilevers will possess piezoelectric actuation allowing for the independent z translation of the probes. The piezoactuators will serve as feedback controls that can control the cantilever movement to maintain a constant electrical conduction measurement. This new SICM-array will then be applied to study the effect of smoking-mediated oxidative stress on the activity of hemichannels. Our laboratory is at the forefront of defining the 3D structure and activity of hemichannels using AFM. Hemichannels play a critical role in maintaining ionic cellular homeostasis and transmitting chemical signals. The activity of hemichannels can be altered through oxidative stress caused by smoking. However, little is known about the distributive and cooperative properties of populations of hemichannels and how they are altered by substance abuse. To probe the effect of oxidative stress on hemichannels, hemichannels will be deposited on custom fabricated nanoporous silicon supports. The support system will be mounted on a two chamber SICM sample holder, in which the top and bottom of the bilayers are electrically separated and ionic current can only pass between the top and bottom chamber through hemichannels. Thus, the conducting SICM-array will be utilized to study the dynamic behavior of hemichannels in multiple locations simultaneously in normal conditions and following exposure to peturbants, such as oxidative agents produced by substance abuse, particularly smoking. Our understanding of drug/substance abuse-mediated hemichannel behavior will help us design preventive and/or therapeutic approaches for substance abuse. The novel SICM-array created in this proposal will have broad applications for the study of electrical signaling and propagation and discovery of novel therapeutics across physiological systems.

Public Health Relevance

Cellular processes in health and disease are guided by the collective coordinated behavior of individual specialized proteins that control chemical and electrical activity known as ion channels. This project creates a novel microscope that examines the coordinated activity of ion channels and how this activity is altered due to the effects of smoking and substance abuse.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31DA034562-02
Application #
8607463
Study Section
Special Emphasis Panel (ZRG1-F04-D (20))
Program Officer
Babecki, Beth
Project Start
2013-01-14
Project End
2016-01-13
Budget Start
2014-01-14
Budget End
2015-01-13
Support Year
2
Fiscal Year
2014
Total Cost
$34,942
Indirect Cost
Name
University of California San Diego
Department
Engineering (All Types)
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Connelly, Laura S; Meckes, Brian; Larkin, Joseph et al. (2014) Graphene nanopore support system for simultaneous high-resolution AFM imaging and conductance measurements. ACS Appl Mater Interfaces 6:5290-6
Kwok, Jeanie; Grogan, Shawn; Meckes, Brian et al. (2014) Atomic force microscopy reveals age-dependent changes in nanomechanical properties of the extracellular matrix of native human menisci: implications for joint degeneration and osteoarthritis. Nanomedicine 10:1777-85
Alfonta, Lital; Meckes, Brian; Amir, Liron et al. (2014) Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy. Anal Chem 86:7674-80
Meckes, Brian; Ambrosi, Cinzia; Barnard, Heather et al. (2014) Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments. Biochemistry 53:7407-14
Meckes, Brian; Arce, Fernando Teran; Connelly, Laura S et al. (2014) Insulated conducting cantilevered nanotips and two-chamber recording system for high resolution ion sensing AFM. Sci Rep 4:4454