Potassium channels play important roles in numerous cellular events in both electrically excitable as well as nonexcitable cells. The molecular basis of how these channels function in membranes is being elucidated by molecular biological and electrophysiological approaches. However little information is known about the mechanisms involved in the regulation and biosynthesis of these membrane proteins. The biosynthesis of potassium channels will be studied in both a cell-free and cell system. Specific potassium channel mRNA will be used to direct protein synthesis in a rabbit reticulocyte lysate system in the presence and absence of rough microsomes. These studies will define the initial post- translation events that take place as the channel is inserted into the rough endoplasmic reticulum. Frog oocytes injected with potassium channel mRNA and neuronally derived tissue will also be used to define further post-translational events that occur as the channel is processed through the Golgi and finally inserted into the plasma membrane. In these studies specific antibodies to potassium channels will be used to identify these biosynthetic intermediates. Immunopurified potassium channels from both the RER and Golgi will be extensively characterized by enzymatic deglycosylation and binding to lectins followed by analysis on different gel systems to determine the extent of post- translational modifications. Pharmacological agents, such as antibiotics, will also be used to define the steps required for transport to the cell surface. In summary, these studies may be expected to outline the biosynthetic history of potassium channels which is an important step in understanding how a cell regulates channel numbers, type, as well as the distribution of these molecules to the cell surface.
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