Development of a Simple Devise to Measure Gap Junction Activity

DESCRIPTION (provided by applicant): Gap junctions are protein channels that conduct ionic current and exchange small molecules between cells. They create functional syncitia in most tissues of the body such as glia, heart, smooth muscle and epithelia. Gap junction malfunction has been implicated in many diseases, but there are no specific reagents to activate or inactivate gap junctions. One of the rate limiting factors in drug development is the tedious nature of current assays. This application proposes to develop a simple tool that applies contemporary microfluidic and electronic technology to an established electrophysiological technique called the sucrose gap. Tissue cultured cells placed on a microfluidic chip are divided electrically in half by a narrow flow of non conducting solution. A four electrode array using phase detection at audio frequencies measures the impedance from one side of the gap to the other. We will also follow the diffusion of fluorescent dyes from one side of the gap to the other simultaneously with the electrical measurement. Analysis of mean current and its variance will provide an estimate of the number of channels, the unitary current and the kinetics of opening and closing. Public Health Relevance: Gap junctions are protein pores between cells that allow them to communicate with ionic currents and chemical messengers. Defects in gap junctions are associated with many diseases including cardiac arrhythmias, cataracts and cancer. This proposal will create a device to screen chemical libraries for drugs that are active on gap junctions.