Molecular mechanisms of the coupling of gating to voltage sensing in transmembrane proteins

Abstract

Voltage gated potassium ion (Kv) channels regulate action potentials of the nervous system by responding to changes in transmembrane voltage, enabling K+ transport across the membrane to restore cells to their resting potential. Comprised of four identical subunits, Kv channels contain four voltage sensing domains arranged on the periphery of a central pore domain. Each voltage sensor is comprised of four transmembrane helices, numbered S1 through S4. The S4 helix, containing four to six highly-conserved, positively-charged arginine or lysine residues, is responsible for voltage sensitivity in Kv channels. The pore domain consists of two transmembrane helices, S5 and S6. The S5 helix constitutes the periphery of the pore domain and is believed to be relatively immobile. The S6 helices, lining the interior of the channel, gate the protein and regulate K+ permeation. Because each subunit of Kv channels contains six transmembrane helices, they are often referred to as 6TM Kv channels. The depolarization of an action potential is initiated as sodium ions enter the cell. At the cellular resting potential of -70 mV, potassium ion channels are closed, and the S4 helix is in its “down” state. As the electrochemical gradient changes, the S4 helices of Kv channels begin to reorient within the membrane. At the peak of the action potential (roughly +20 mV), the S4 helices exist in their “up” state. This conformational transition of the S4 helix is coupled to the pore domain via the S4-S5 linker, a short, amphipathic helix along the intracellular membrane-water interface. By bridging the C-terminus of the voltage sensor to the N-terminus of the pore domain, the S4-S5 linker couples the voltage sensitivity of the voltage sensor to K+ conduction in the pore domain. Because they begin opening at voltages less than 0 mV, all crystal structures of Kv channels contain an open pore domain. With no structure in the closed conformation, the mechanism of gating in Kv channels remains unclear. Nevertheless, significant biophysical studies have revealed insights into both the closed conformation and the gating transition itself. In this dissertation, I will explore questions relevant to the gating mechanism in voltage gated potassium ion channels through fully-atomistic molecular dynamics (MD) simulations. First, in Chapter 2, I will address the potential role of the 310 helical conformation found in the C-terminal end of S4 in the crystal structures of Kv channels. Spanning eight or more residues, these 310 helices are both uncharacteristically long and conserved in K+ channel crystal structures. By simulating the Kv1.2/2.1 chimera channel’s voltage sensor embedded in a lipid bilayer, I find that an alpha to 310 helical interconversion of the S4 helix reproduces many experimental measurements of the open and closed states of Kv channels. In Chapter 3, I perform molecular dynamics simulations of the entire Kv1.2/2.1 chimera channel. First, I examine the impact of an alpha to 310 helical interconversion of the S4 helix on the pore domain of the channel. Though the results are consistent with the results in Chapter 2 (and the corresponding experimental measurements), I find that this secondary structural modification is insufficient to influence the pore domain of the channel on the timescale of my simulations. In the second half of Chapter 3, I use molecular dynamics simulations to generate a closed state model of the Kv1.2/2.1 chimera from luminescence resonance energy transfer (LRET) measurements of the closed conformation of KvAP. The resulting structure is indeed closed, and also recapitulates a number of experimentally determined measurements of the closed channel. In Chapter 4, I focus on the pore domain. First, using targeted molecular dynamics simulations, I generate a transition between a closed model of the KvAP linker and pore domain to the open conformation. Then, using an umbrella sampling method, I quantify the energetics of the gating transition in KvAP and assess the physiological implications. In agreement with experimental studies of Kv channel energetics, I find that the open pore is roughly 2.7 kcal/mol lower in free energy than the closed conformation. The targeted molecular dynamics and umbrella sampling simulations reveal additional insights into the gating mechanism of KvAP. Lastly, in Chapter 5, I use MD simulations to gain insights into the binding mechanism of VSTx1, a Kv channel inhibitor. By using the experimentally determined neutron scattering density profile of the VSTx1 toxin bound to a lipid bilayer as a restraint for molecular dynamics simulations, I recreate the experimental scattering density profile, and also offer insight into the binding of VSTx1 to a lipid membrane.