Hadley, Jennifer Kathleen; (2000) Identification of channels underlying the M-like potassium current in NG108-15 neuroblastoma-glioma cells. Doctoral thesis (M.Phil.), University College London.

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Abstract

NG108-15 cells express a potassium current resembling the M-current found in sympathetic ganglia. I contributed to the identification of the channels underlying this NG108-15 current. I used patch-clamp methodology to characterise the kinetics and pharmacology of the M-like current and of three candidate channel genes, all capable of producing "delayed rectifier" currents, expressed in mammalian cells. I studied two Kv1.2 clones: NGK1 (rat Kv1.2) expressed in mouse fibroblasts, and MK2 (mouse brain Kv1.2) expressed in Chinese hamster ovary (CHO) cells. Kv1.2 showed relatively positive activation that shifted negatively on repeated activation, some inactivation, block by dendrotoxin and various cations, and activation by niflumic acid. It had persuasive differences from the M-like current. mErg is the mouse homologue of the cardiac ether-à-go-go-related gene channel HERG. Having discovered that the HERG blocker WAY-123,398 partially inhibited the M-like current, I studied mErg1a in CHO cells. mErg1a, as previously reported, undergoes intense inactivation, removed on hyperpolarisation to give a transient increase in current preceding deactivation. I report that it can also produce a sustained current with kinetic and pharmacological correspondences to the slow component of the M-like current. Blocking the presumed mErg component in NG108-15 left a current very similar to the sympathetic ganglion M-current. The latter was recently suggested to comprise a heteromultimer of KCNQ2 and KCNQ3, two neuronal relatives of the cardiac KvLQT1. I expressed KCNQ2 and KCNQ3 in CHO cells, separately and together. Their kinetics match those of the M-current. The M-current blocker linopirdine blocks these channels indiscriminately, while tetraethylammonium blocks KCNQ2 > KCNQ2/KCNQ3 > KCNQ3. Since the selective blockers linopirdine and WAY-123,398, applied together, eliminate the total M-like current, Kv1.2 and other channels are unlikely to contribute. I conclude that blockers, when backed up by kinetic and expression data, were a particularly valuable tool for identifying M-like current components.

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