Novel effects of glycosylation loss and anaesthetics on Kv1.2 channels

• Author / Creator

  Fajonyomi, Daniel Oluwamayowa

• Slow gating regulation is an extrinsic regulatory mechanism that inhibits the activation of Kv1.2 channels. Physiological factors modulating slow gating regulation are incompletely understood and the interplay between slow gating regulation and Kv1.2 channel pharmacology is unexplored. In chapter 3, I investigated how N-glycosylation influences Kv1.2 sensitivity to slow gating regulation. Using site-directed mutagenesis, I generated glycosylation deficient Kv1.2 channels by mutating the Kv1.2 N-glycosylation site at asparagine (N) 207. Using whole-cell patch-clamp electrophysiology, I characterized the activation properties of glycosylation deficient Kv1.2 channels in mammalian cell lines. I found the activation of glycosylation deficient Kv1.2 channels was strikingly inhibited compared to wild-type Kv1.2, and further investigated whether this arose from direct effects on channel gating, or an interaction with Kv1.2 slow gating. Manipulations that attenuate slow gating regulation relieved the inhibited activation of these channels. Specifically, the Thr252Arg mutation, previously shown to attenuate slow gating regulation, also weakened the slow gating features of glycosylation deficient Kv1.2 channels. Activating prepulses caused accelerated activation of glycosylation deficient channels, consistent with activity-dependent relief of slow gating. Furthermore, co-expression with a recently described Kv1.2 regulatory partner, Slc7a5, abolished slow gating regulation in both WT and glycosylation deficient Kv1.2. Finally, I showed glycosylation deficient channels and WT Kv1.2 channels exhibit similar activation properties in Xenopus oocytes using two-electrode voltage clamp, in contrast to the apparent differences between these channels in mammalian cell lines, illustrating that the inhibited activation of glycosylation deficient channels is likely not an intrinsic property. Taken together, these results demonstrate that glycosylation deficient Kv1.2 exhibits enhanced sensitivity to slow gating regulation.

  I also investigated the influence of slow gating regulation on Kv1.2 pharmacology by testing the response of various Kv1.2 mutants to the general anaesthetic propofol. Propofol modulation of Kv1.2 mutants was measured using whole-cell patch clamp electrophysiology of cell lines with intact mechanisms of slow gating regulation. Propofol exerted a combination of inhibitory and potentiating effects on Kv1.2 channels. Propofol inhibited Kv1.2 current magnitudes and caused a time-dependent decay of Kv1.2 currents consistent with open state block of Kv1.2 channels. However, Kv1.2 channel mutants showed differing sensitivities to these inhibitory effects, correlated to their propensity for slow gating regulation. Superimposed on these inhibitory effects, propofol shifted the voltage-dependence of Kv1.2 to more negative voltages and accelerated Kv1.2 channel activation kinetics. These findings suggest these potentiating effects are due to propofol-mediated disruption of the slow gating regulation of Kv1.2 channels. Overall, this thesis demonstrates that post-translational modifications of Kv1.2 can influence their sensitivity to extrinsic regulatory mechanisms, highlight the impact of extrinsic regulatory mechanisms on ion channel pharmacology, and describe the first instance of modulation of slow gating regulation of Kv1.2 by a small molecule (propofol).

• Subjects / Keywords

  • Kv1.2
  • Propofol
  • Glycosylation
  • Slc7a5
  • Slow gating regulation
  • Use-dependent activation

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-9v1a-xf24

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.