A Microfluidic Assay for Measuring Electrical Conductivity of Gap Junction Channels
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A Microfluidic Assay for Measuring Electrical Conductivity of Gap Junction Channels Cedric Bathany1, Frederick Sachs2 and Susan Z. Hua1,2 1
Department of Mechanical and Aerospace Engineering, SUNY-Buffalo, Buffalo, NY 14260, U.S.A. 2
Department of Physiology and Biophysics, SUNY –Buffalo, Buffalo, NY 14260, U.S.A.
ABSTRACT We have developed a microfluidic sensor to measure the electrical coupling through gap junctions across a 2D sheet of cultured Normal Rat Kidney cells. The chip is based on a tristream laminar flow with conductive solutions on either end and a nonconductive solution in the middle stream. When an electrical voltage is applied, the current can only pass through the cell sheet, thereby enabling us to measure the electrical coupling of gap junctions between cells. Using this sensor we have measured the effect of 2-aminoethoxydiphenyl borate (2-APB) on Cx43 channels. We show that 2-APB reversibly inhibits electrical coupling of Cx43 in NRK cells. Moreover, we screened other potential candidates to block Cx43, 1-Heptanol and GsMTx4, and examined their effects on Cx43 gap junctions. INTRODUCTION A vast number of biological processes rely on the ability for cells to communicate with each other through exchange of ions and molecules. This intercellular communication between the neighboring cells is mainly accomplished by hexameric channels, named gap junctions [1]. Most cells are known to express multiple connexins, and there are no well identified blocking reagents for a specific type of junction channel. Clearly, there is a need for identifying chemical reagents that inhibit specific gap junctions and understanding their effectiveness on the modulation of gap junction activities. Several chemical agents, such as 1-Heptanol and 1-Octanol, are known as non-selective gap junction blockers that directly interact with gap junctions by modifying the plasma membrane fluidity. Other reagents, such as 2-APB, have been reported to show different blockage mechanisms to selected connexins, such as Cx43 and Cx50 [2]. In this regard, it remains challenging to recognize a perfect candidate to specifically block the gap junction. Therefore, there is a need to screen chemical agents, such as the peptide GsMTx-4, an ion channel blocker, that interact with cell membrane. Microfluidic techniques offer a promising method to study cellular activities with high throughput [3]. Specifically, microfluidic chips can provide a flexible and easy-to-use design to screen chemical reagents for specific gap junctions [4]. We have developed a microfluidic sensor capable of recording electrical coupling of gap junctions in cultured 2D cell sheets. The microfluidic design involves a tri-stream laminar flow with two conducting solutions on either
sides and a sucrose based non-conducting solution in the middle, forming a sucrose gap [5]. Cells were submerged across the three streams, and the ionic transport from cell to cell across the non-conductive region was measured by applying a voltage across the non-conductive gap. The gap junct
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