Molecular characterization of ATP-sensitive potassium channels

K_ATP channels are regulators of a variety of physiological functions, including insulin secretion, blood pressure, and response of the heart and brain to ischemia. Sulfonylurea receptors (SURx) are multi-spanning transmembrane proteins of the ATP-binding cassette (ABC) family, which associate with Kir6.x to form ATP-sensitive potassium channels (K_ATP). My studies focus on three aspects of K_ATP channel structure and function: the transmembrane topology of SUR1, affects of a SUR1 genetic mutation, and the role of oligosaccharides in K_ATP trafficking.; Two models, with 13–17 transmembrane segments, have been proposed for SURx topologies. Investigation of SUR1 using a labeling surface biochemical assay was consistent with a 17-transmembrane model, and revealed that putative external loops 1–3, 5–6, 8 and N-terminus are external, while putative internal loops 3–8 and C-terminus are internal. Immunofluorescence microscopy was used to localize epitopes at putative external loops 3 and internal loops 5 and 7. These results support the 17-transmembrane model with two pairs of transmembrane segments as possible reentrant loops.; Appropriate assembly and protein trafficking of K_ATP channels is essential for accurate insulin secretion. Persistent hyperinsulinemic hypoglycemia of infancy, caused by deletion of phenylalanine 1388, SUR1 ΔF1388, was shown to result in defective trafficking to the plasma membrane and diminished glycosylation when co-expressed with Kir6.2. In addition, we demonstrated a decrease in channel currents, ATP/ADP sensitivity, and glycosylation of SUR1_AAA ΔF1388 when co-expressed with Kir6.2 compared to wild type SUR1.; Oligosaccharides play a role in trafficking and folding of many membrane proteins. I showed a significant decrease in surface expression of SUR1 single or double glycosylation site mutants (N10Q, N1050Q) when co-expressed with Kir6.2. Additionally, retention within the ER of the SUR1 double glycosylation mutant was revealed under the same conditions. Mutation of the ER retention signal partially restored surface expression of the SUR1 double glycosylation mutant. Furthermore, facilitation of SUR1 surface expression and glycosylation by coexpression with Kir6.2 was confirmed. These studies suggest that SUR1 glycosylation is important for the proper trafficking and surface expression of K_ATP channels.; These investigations advance our understanding of K_ATP and its role in disease and normal physiology.