Giblin, Jonathan Peter; (2001) An investigation of the biochemical interaction between subunits of the ATP-sensitive potassium channel. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

ATP sensitive potassium channels (KATP) are an octameric assembly of four pore forming Kir6.0 and four Sulphonylurea receptor (SUR1, SUR2A or SUR2B) subunits. They form a link between cellular metabolism and electrical excitability in a wide range of tissues. The central questions addressed in the thesis relate to how these subunits can physically interact with each other to form the channel. Cloned subunits of the channel subunits have been used to express the proteins in heterologous systems. Monoclonal stable cell lines have been produced in HEK293 cells expressing different combinations of subunits. These lines were used in the characterisation of rabbit polyclonal antisera raised to peptides corresponding to various domains on channel subunits. It was shown by co-immunoprecipitation that KATP channel subunits physically associate. Chimeras constructed between Kir6.2 and a related protein Kir2.1 co-expressed in monoclonal stable lines with an epitope-tagged SUR1 were used in a co-immunoprecipitation experiments to show that a region in the proximal C-terminus of Kir6.2 is important for biochemical interaction with SUR1. An attempt to determine regions on SUR1 responsible for interaction with Kir6.2 was made using C-terminal deletion mutants of SUR1 co-expressed with Kir6.2 in monoclonal stable lines. Co-immunoprecipitation experiments showed that the C-terminus of SUR1 was not necessary for physical interaction with Kir6.2. The effect of the C-terminal deletions of SUR1 on the trafficking of SUR1 and Kir6.2 to the cell membrane was also studied using immunofluorescence microscopy. The possibility of heteromultimerisation between different subtypes of KATP channel subunits was also investigated using stable lines co-expressing Kir6.1 with Kir6.2 and SUR1 with SUR2A. Co-immunoprecipitation experiments indicated that physical interaction can occur between the different Kir6.0 subunit subtypes but not the SUR subtypes.

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