Single glucose molecule transport process revealed by force tracing and molecular dynamics simulations

Transporting individual molecules across cell membranes is a fundamental process in cellular metabolism. Although the crystal diffraction technique has greatly contributed to our understanding of the structures of the involved transporters, a description of the dynamic transport mechanism at the single-molecule level has been extremely elusive. In this study, we applied atomic force microscopy (AFM)-based force tracing to directly monitor the transport of a single molecule, d-glucose, across living cell membranes. Our results show that the force to transport a single molecule of d-glucose across cell membranes is 37 ± 9 pN, and the corresponding transport interval is approximately 20 ms, while the average speed is approximately 0.3 μm s^-1. Furthermore, our calculated force profile from molecular dynamics simulations showed quantitatively good agreement with the force tracing observation and revealed detailed information regarding the glucose transport path, indicating that two salt bridges, K38/E299 and K300/E426, play critical roles during glucose transport across glucose transporter 1 (GLUT1). This role was further verified using biological experiments that disrupted these two bridges and measured the uptake of glucose into the cells. Our approaches led to the first unambiguous description of the glucose transport process across cell membranes at the single-molecule level and demonstrated the biological importance of the two salt bridges for transporting glucose across GLUT1.