Hillman, D.J.; (2005) Membrane currents evoked by vasoactive compounds in vascular endothelial cells: Contributions of small and intermediate conductance calcium-activated potassium channels. Doctoral thesis , University of London.

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Abstract

The contribution of different calcium-activated potassium channel subtypes to agonist-evoked whole-cell currents was studied in cultured pig coronary artery endothelial cells. From a resting membrane potential of-5.9 0.5mV (n=102), 1-lOuM ATP, 1-1 OnM substance P and 1-lOOnM bradykinin hyperpolarised cell rafts to -50.7 1.6mV (n=76), -45.7 4.7mV (n=19) and -59.1 3.5mV (n=16), respectively. In small clusters of cells, 1 OuM ATP evoked outward currents which reversed close to EK and were sensitive to both the SKca channel blocker UCL 1848 (IC5o 1.2nM -65% maximal block) and the IKca/BKca channel blocker charybdotoxin (-85% block at 30-100nM). Surprisingly lOuM clotrimazole, a non selective blocker of IKca channels, abolished ATP-evoked currents in a total of three out of five cells. This requires further study. ImM 1-EBIO, which increases the calcium sensitivity of SKca and IKca channels, activated currents which were sensitive to lOOnM UCL 1848 and luM clotrimazole (blocked by 57.0 15.1% (n=3) and 89.0 1.6% (n=4), respectively). When applied in combination, these two blockers essentially abolished 1-EBIO evoked currents. Buffering intracellular calcium to 1.5uM activated outward currents which were sensitive to lOOnM UCL 1848, lOOnM charybdotoxin and lu.M clotrimazole (blocked by 28.3 5.4% (n=27), 101.2 0.5% (n=3), and 82.6 3.7% (n=22), respectively. Plasma membrane delimited expression of the SK3 channel protein was detected using fluorescence immunohistochemistry. In many vessels endothelium-derived hyperpolarising factor (EDHF)- mediated vasodilation is abolished by a combination of SKca and IKca channel blockers, which are frequently ineffective when applied alone. This has led some to suggest the existence of a novel channel with unusual pharmacology. The present study demonstrates, however, that separate SKca and IKca channels contribute to endothelial cell currents underlying the EDHF pathway. Based on protein expression and UCL 1848-sensitivity it is further proposed that the contributing SKca channels are formed of SK3 subunits.

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