Secretion of hormones and neurotransmitters is critical for biological function in higher organisms. Endocrine hormones and neurotransmitters are stored in intracellular vesicles and released via exocytosis. The benefits, as well as the challenges, of single cell analysis have been discussed extensively. Briefly, detection of exocytotic release requires high temporal (ms) and spatial (m) resolution, as well as high (M-nM) sensitivity, and enhanced selectivity when multiple secretory products are released, e.g. from chromaffin or mast cells. The application of carbon fiber microelectrodes (CFMEs) to detection of individual exocytosis events at the single cell level has provided key information regarding the regulation and spatial organization of neurotransmission, as well as validated long standing hypothesis for glucose-stimulated insulin secretion and revealed new signal pathways. While powerful, detection using CFMEs is limited to electroactive species and the spatial resolution of electrochemical imaging is further limited by the size of the probe, the resolution/precision of electrode placement, and the distance between the probe and cell surface. Because of these and other limitations, highly spatially resolved maps of secretion have not been obtained for endocrine hormone release. In this project, we describe new methods to allow the study of chemical/biochemical efflux from single cells at subcellular spatial resolution. Specifically, we will develop Ion Channel Probes (ICPs) which consist of a pipette with a stabilized lipid bilayer at the tip. Insertion of an ion channel into the bilayer results in a bio/chemical sensor based on the transport properties of the ion channel. When combined with Scanning Ion Conductance Microscopy (SICM), precision distance control is achieved and a new tool - Ion Channel Probe - Scanning Ion Conductance Microscopy (ICP-SICM) for single cell studies is realized.
Work described in this proposal will develop new methods that can ultimately be used to understand regulation of insulin and glucose levels, at the level of single cells. The spatial distribution of active release sites in the cell membranes of ?-cells an ?-cells, and the ability to measure chemical release with high spatial precision while controlling the molecular environment in the immediate vicinity are key steps towards understanding the roles that spatial organization of exocytotic release within pancreatic islets exert on islet functon and ultimately blood glucose homeostasis in normal and disease states.
| Shi, Wenqing; Zeng, Yuhan; Zhu, Cheng et al. (2018) Characterization of Membrane Patch-Ion Channel Probes for Scanning Ion Conductance Microscopy. Small 14:e1702945 |
| Agasid, Mark T; Wang, Xuemin; Huang, Yiding et al. (2018) Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells. Protein Expr Purif 146:61-68 |
| Shi, Wenqing; Friedman, Alicia K; Baker, Lane A (2017) Nanopore Sensing. Anal Chem 89:157-188 |
