Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.634577
Title: The mechanisms of metabolic regulation of the cloned equivalent of the vascular Katp/Kndp channel
Author: Tabasum, F.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2007
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Abstract:
At the molecular level, Katp is an octameric protein complex composed of an inwardly rectifying K+ channel subunit (Kir6.*) which forms the channel pore and a regulatory sulphonylurea receptor subunit (SUR). By sensing intracellular nucleotide concentrations, Katp channels couple the membrane potassium conductance of a cell to its metabolic state. To understand the molecular basis of metabolic regulation of the Katp/Kndp channel Kir6.1/SUR2B, the cell-based Rubidium-86 (86Rb+) efflux assay was employed, using HEK293 cells as the expression system. 86Rb+ efflux was activated on the addition of lOpM levcromakilim and metabolic poisoning (induced by 20mM 2-deoxyglucose and 2.5mM sodium cyanide), and inhibited by lOpM glibenclamide. The subsequent 86Rb+ distribution between intracellular and extracellular space was determined via the measurement of Cherenkov radiation, the relative amount of 86Rb+ in the cell supernatant being a direct measure of channel activity. The functionality of selected mutants was also investigated under physiological conditions by the perforated patch- clamp method, whereby currents were evoked via voltage clamp recordings over 1000ms voltage steps between -150mV and +50mV from a holding potential of-80mV. AN-Kir6.1-AC truncations were made in an attempt to understand the role of the pore- forming subunit pharmacologically in channel gating, as has previously been shown for Kir6.2AC26. Mutants for which the RXR motif was removed were surface expressed (as shown via irnmunofluorescent staining using an anti-HA-fluorescein conjugated antibody), and showed increased basal efflux in the absence of SUR2B which was reduced by inhibitors which bind to the pore-forming subunit (lmM BaCh or lOOpM PNU-37883A). However, these mutants did not display intrinsic ATP sensitivity, as for AN-K.ir6.2-AC, and for Kir6.1AC48/AN13 this was confirmed by perforated patch clamping. When Kir6.1AC48/AN13 was expressed with SUR2B, glibenclamide was able to reverse efflux induced on metabolic inhibition, but it was suspected that under this condition the sterical conformation of the channel changed which prevented Kir6.*-specific inhibitors from binding (since they were not able to reverse metabolically induced efflux). In contrast, application of either ImM BaCb or lOOuM PNU-37883A was able to reverse metabolically induced efflux for Kir6.2AC26/SUR2B. On the introduction of mutations into NBD1 or NBD2 (K708A and K1349M respectively) of SUR2B, Kir6.1/SUR2B K708A/K1349M was shown to be non-functional, whereas Kir6.2/SUR2B K708A/K1349M displayed normal pharmacology on the addition of KCOs and metabolic inhibitors. In conclusion, Kir6.1/SUR2B is sensitive to metabolic poisoning in an analogous fashion to Kir6.2/SUR2B. but the Kir6.1 pore-forming subunit does not display intrinsic metabolic sensitivity, unlike Kir6.2. Metabolic sensitivity of Kir6.1/SUR2B is determined by both nucleotide binding domains of SUR2B, and finally the pharmacology of metabolically attenuated currents is different from those activated by a Katp channel opener.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.634577  DOI: Not available
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