In this thesis it is demonstrated that leptin and insulin hyperpolarise hypothalamic glucose responsive neurones via the activation of the large conductance ATP-sensitive (K<sub>APT</sub>) channel. This channel was potassium selective, had a single channel conductance of 150 pS and channel activity was inhibited by micromolar tolbutamide and millimolar internal ATP. Brain cortical cell bodies and nerve terminals possess a large-conductance calcium-activated potassium (BK) channel. The nerve terminal BK channel switched from high to low activity modes, whereas cell body BK channel activity inactivates during depolarisation. Furthermore, BK channel inactivation was abolished by internal trypsin treatment, suggesting an inactivating particle was associated with the channel. Internal application of alkaline phosphatase irreversibly removed mode switching and inactivation of cortical BK channels. Blocking the cell body BK channel pore with 100 mM intracellular tetraethylammonium (TEA) prevented alkaline phosphatase removal of inactivation, indicating that the phosphatase site of action was located close to the pore. Finally, protein kinase A (PKA) increased the occurrence of the high BK channel activity mode whereas PKA retarded the full recovery of BK inactivation induced by hyperpolarisation. In a separate study it was demonstrated that stably expressed human brain BK (<I>hSlo</I>) channels inactivate in a trypsin-insensitive manner. This inactivation was not due to barium contamination, since 5 μM internal barium blocked <I>hSlo</I> channels only during strong depolarisations, yet inactivation was observed at less positive potentials. Furthermore co-expression of either <I>hSlo</I>β-1, or voltage-gated potassium (Kv) β1.1 or β2.1 subunits with the <I>hSlo</I> α-subunit did not affect the extent or rate of channel inactivation. Finally, the Kvβ-subunits moved the calcium and voltage curves of <I>hSlo</I> to more negative voltages and altered the activation and deactivation kinetics in a manner almost identical to that observed on co-expression of <I>hSlo</I>β-1 subunit with <I>hSlo</I> or by increasing the internal calcium concentration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:322636 |
| Date | January 1999 |
| Creators | Smith, Mark Allan |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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