Visualizing functional ion channels at the cell surface

Irving HR1,2, Abad I2, Nguyen D-T2 and Manallack DT2

  1. La Trobe Institute for Molecular Science, La Trobe University Bendigo VIC 3550.
  2. Monash Institute of Pharmaceutical Sciences, Monash University Parkville VIC 3052.

Assembly of components of ion channels into functional heteromers at the cell surface is still mysterious. To begin to obtain clues about this process we use HTR3A and HTR3C receptor subunits that have each been tagged with separate fluorescent proteins in the second intracellular loop. The subunits are transiently expressed in HEK293T cells and visualised by high resolution microscopy. These subunits are processed normally through endoplasmic reticulum and Golgi pathways to reach the plasma membrane surface. Using TIRF microscopy we show that the subunit heteromers are formed intracellularly. Whole cell patch clamp reveals that the subunits are functional at the surface where they respond to 5-HT and this response is suppressed by ondansetron. Analysis of our TIRF images has allowed us to determine the proportion of different heteromers within the cell and at the surface. This finding is important as it will allow a more precise analysis of how different drugs contribute to subtle changes in heteromer versus homomer function that in turn may be related to individual receptor components.