Study of Arabidopsis thaliana COPT1 Transporter Function Using a Plant Membrane on a Chip Platform

A growing interest has been placed on the development of experimental techniques to understand the response of plants to climate change and stressors. The study of membrane proteins and transporters is integral towards understanding the key roles and functions of cellular processes in plant stressor response. Traditional techniques for investigating protein biophysics, such as the patch-clamp method, are less than perfect, lacking generalizability, ease-of-use, and speed. We propose a novel, fast, and sensitive biomimetic device for examining transport protein behavior based on a supported lipid bilayer (SLB). This platform comprises native plant membrane material on a transparent and conductive polymer (PEDOT:PSS), part of an organic electrochemical transistor (OECT). This allows the device to be amenable to a variety of optical and electrical characterization techniques. We have leveraged this platform to investigate the transport characteristics of a well-conserved copper (Cu⁺) transporter protein COPT1 in Arabidopsis thaliana, responsible for maintaining copper homeostasis in plants. Mesophyll protoplast transiently expressing GFP and overexpressed COPT1:GFP were induced to form microvesicles. These microvesicles were reconstituted as a SLB using lipid-vesicles fusion on top of PEDOT:PSS. The formation and quality of the SLB was characterized by fluorescence recovery after photobleaching (FRAP) and confirmed by an increase in system impedance using Electrical Impedance Spectroscopy (EIS). Transport of Cu⁺ through COPT-1 SLBs were measured by EIS under different concentrations 0-100 µM. In the presence of Cu⁺, COPT1 opens, allowing the transport of ions through the membrane, resulting in a decrease in the system impedance. Without Cu⁺, the system impedance returns to the baseline SLB level pointing to system Cu⁺ sensitivity. This indicates that our platform has the potential to be used to investigate plant transporters and proteins involved in response to plant stressors.