@article{Intracellular:4776,
      recid = {4776},
      author = {Koh, Young Hoon},
      title = {C-Type Inactivation and Voltage Sensor to Intracellular  Gate Coupling in Eukaryotic Potassium Channels},
      publisher = {University of Chicago},
      school = {Ph.D.},
      address = {2022-08},
      pages = {106},
      abstract = {Abstract for Chapter 2:The structural basis for C-type  inactivation in eukaryotic voltage-gated channels is still  unknown and unclear at best. This is partially because the  kinetics and depth of C-type inactivation in eukaryotic  channels tend to be slower and shallower than in  prokaryotic channels such as KcsA. In other words, each  channel at 0 mV is more likely to be in a conductive  conformation, and thus, trapping the protein in the  inactivated state is quite difficult. Despite much effort  from the field over the past decade or so, capturing this  elusive C-type inactivated state structurally has been a  great challenge. To obtain the C-type inactivated state in  Kv1.2-2.1, then, we decided to look back towards KcsA. In  KcsA, the C-type inactivation is characterized by a  constricted or pinched conformation in the selectivity  filter, which prevents ion conduction. The Mackinnon lab  captured this state by depleting the potassium ion from the  protein and thus introducing a microenvironment near the  selectivity filter that would favor the constricted  inactivated state (1k4d). Applying the same idea to  Kv1.2-2.1 to capture its inactivated state is a natural  extension of this strategy. Not only is this an already  proven way to induce conformational changes in the filter,  but also it may be a more natural way to induce  inactivation compared to introducing a highly disruptive  mutation. After all, the C-type inactivated or  non-conducting filter is by its very definition one that is  depleted of K+ ions. Therefore, creating the environment  around the channel to naturally induce its conformational  change may give us the best chance to observe a  conformational change that is associated with the native  C-type inactivated state. In this chapter, I will discuss  the arduous challenges I faced trying to stabilize the Kv  chimera protein in various low K+ environments with various  counterions to K+. I will also discuss how I solved this  issue and ultimately obtained the structures and function  of Kv1.2-2.1 in low K+. 

Abstract for Chapter  3:Voltage-activated potassium (Kv) channels play a critical  role in propagating action potentials within cells by  opening in response to membrane depolarization and closing  in response to hyperpolarization. The proper and timely  manner that these channels open and close is essential for  the health of the entire organism. Therefore, it is  worthwhile studying how these channels transition from the  open to closed state and vice-versa. On a deeper level,  understanding how the movement of the voltage sensing  domain triggered by the membrane electric field affects the  opening and closing of the intracellular gate in  domain-swapped, depolarization-activated channels such as  Shaker and Kv.1.2 is of particular interest. Since the  determination of its crystal structure in 2005, Kv1.2 (or  Kv1.2-2.1) has been the model channel for understanding the  mechanistic questions of Kv channels at the atomic level.  Over the past two decades, several structures of Kv1.2 WT  and mutants in various detergent or lipid environments have  been determined. One significant common factor among all of  the experimental structures of Kv1.2 (or Kv1.2-2.1) is that  they display an open intracellular gate. In other words, we  have yet to obtain the experimental structure of a  voltage-activated potassium channel in its closed state.  Here, in this Chapter, we present a 3.4 Å cryo-EM structure  of Kv1.2-2.1 in detergent DDM/CHS with the mutation ILT in  the S4 helix. This mutant has been shown functionally to  right-shift the conductance-voltage (G-V) curve and  decrease the opening probability (P0). Although we did not  capture a channel with a closed gate as we anticipated, the  experimental structure displays some significant  conformational changes in the voltage sensor that seems to  indicate that the voltage sensor has become decoupled from  the pore as a result of the ILT mutations. In the  structure, the voltage sensor splays outward and some  critical electrostatic interactions between gating charges  and their countercharges are lost while new ones are  formed. We believe the conformation captured by our  experimental structure corresponds to a transitional state  from the closed to the opened gate, as the voltage sensor  moves in accordance to changes in voltage in its native  state.
},
      url = {http://knowledge.uchicago.edu/record/4776},
      doi = {https://doi.org/10.6082/uchicago.4776},
}