This competing renewal focuses on one fundamental question: the relationship between macroscopic transport phenomena and molecular events. During the past decade new techniques such as patch clamp, amperometry, and fluorescent microscopy have provided a wealth of information about co-transporters. Yet uncertainly exists about what these new data mean and how they come together with the existing literature on co-transporters. To get to the bottom of these issues we will compare transporters in their dual function as carriers and channels at both the macroscopic and microscopic level.
Specific Aim I combines classical uptake and binding techniques with new data using fluorescent substrates. Our goal is to resolve binding and transport in real time at the sub-cellular level.
Specific Aim II correlates whole-cell and patch-clamp electrophysiology and compares these data with cryo-EM images of transporters. We use mammalian cells for these first two aims, and take advantage of inducible cell lines and gain-of-function and loss-of-function mutants.
Specific Aim III uses frog oocytes in the whole cell and cut-open configuration to test mechanistic predictions of co-transport arising from the first two aims. Frog oocytes are suitable for simultaneous radioligand-uptake and two-microelectrode voltage clamp analysis. In the cut-open oocyte preparation, simultaneous voltage clamp with internal and external solution exchange are possible. This proposal thus emphasizes new experimental protocols in combination with traditional approaches to focus on transporter biophysics at the molecular level. ? ?
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