UBC Theses and Dissertations
Structural and genetic modulators of voltage-gated potassium channel activation kinetics Rezazadeh-Roudsari, Saman
Voltage-gated potassium (Kv) channels regulate membrane excitability and are therefore critical determinants of cellular function. However, the detailed mechanisms by which Kv channel activity is modulated are not well understood. This thesis investigates the modulation of activation of Kv1.2 and KCNQ1 channels. These studies reveal that Kv1.2 can activate via two different pathways that produce two distinct gating phenotypes/modes. In the 'slow' gating mode, the activation V 1/2 was shifted by +30 mV and activation kinetics were at least 20-fold slower than those of channels gating through the 'fast' mode. This offers an explanation for the wide variations in the reported activation kinetics of Kv1.2 in the literature. Introduction of a positive charge at or around threonine 252 (T252) in the S2-S3 cytoplasmic linker of Kv1.2 trapped channels in the 'fast' activation mode, suggesting that this region may act as the molecular switch in Kv1.2. Consistent with this, the S2-S3 linker was shown to mediate the gating-modifying effect of a mutation (T46V) in the cytoplasmic T1 domain of Kv1.2. Excision of patches containing Kv1.2 also trapped channels in the 'fast' gating mode, indicating cytoplasmic regulators may also modify the gating mode via the S2-S3 linker. We have ruled out cytoplasmic regulation by PIP₂, polyamines and phoshporylation. Interestingly, one kinase inhibitor, KN-93, a commonly used calcium/calmodulin-dependent protein kinase II inhibitor, was found to be a direct extracellular blocker of many different Kv channels including Kv1.2. Finally, a novel missense mutation at the intracellular end of the S3 helix in a mutant KCNQ1 channel (V205M), detected in an aboriginal community with a high prevalence of long QT syndrome and sudden death, was shown to cause a depolarizing shift in the voltage dependence of activation and a slowing of activation kinetics. This resulted in reduced repolarization reserve during the cardiac action potential and a likely increased susceptibility to the initiation of arrhythmias. The close positioning of this mutation to the S2-S3 linker provides a putative structural working model for the gating switch in Kv1.2 that involves changes in the hydrophobic packing of the S3 helix and its influence on S4 voltage-sensor movement.