The Merger of Synthetic Biology with Bioelectronics Enables Study of Hard-to-Reach Organelle Ion Channel Proteins

The transport of ions through ion channel proteins has traditionally been characterized using patch clamp techniques where a small patch of a cell’s plasma membrane expressing an ion channel of interest is sealed across an electrode. When the ion channel of interest is not natively expressed in the plasma membrane, modifications to the gene must be made to drive its expression there, which changes the protein and its native membrane environment and can potentially alter its native function. However, ion channels in organelles play important roles in cell homeostasis, for example, TMEM165 resides in the Golgi Apparatus in humans and mutations of this channel have been tied to Congenital Disorders of Glycosylation (CDG). CDG is a family of metabolic diseases affecting the glycosylation pathway, which is dependent on the regulation of ion concentrations in the Golgi Apparatus. TMEM165, and its yeast ortholog GDT1, are implicated as cation transporters of calcium and manganese, and in regulating pH homeostasis in the Golgi, making them highly relevant to human health. Disruptions in TMEM165 function lead to glycosylation abnormalities, causing CDG, and highlight the need for effective therapeutic strategies. A limiting factor in the development of therapeutics that target these proteins in their inaccessibility and challenging reconstitution into functional assays. In this work, we take another approach. We demonstrate the cell-free synthesis and integration of TMEM165 and GDT1 into two functional assays. First, these proteins are synthesized into fluorescent vesicles for optical readout and confirmation of function, second, supported lipid bilayers coating microelectrode arrays for electrical assessment of ion transport. Using these bioelectronic devices, we characterize calcium and manganese ion transport of these proteins and of mutants that have been clinically observed in CDG patients. We find that protein sequence point mutations in the channel wall impact ion transport, correlating to the observed phenotype. With this new platform, screening drugs to counteract these mutants opens an avenue to the development of therapies that could mitigate the impacts of this disease.