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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.