Activation of the intrinsic aqueous water-wire proton conductance (GAQ) in Hv1 channels is controlled by changes in membrane potential and the transmembrane pH gradient (ΔpH). The mechanism by which changes in ΔpH affect the apparent voltage dependence of GAQ activation is not understood. In order to measure voltage sensor (VS) activation in Hv1, we mutated a conserved Arg residue in the fourth helical segment (S4) to His and measured H+ currents under whole-cell voltage clamp in transfected HEK-293 cells. Consistent with previous studies in VS domain containing proteins, we find that Hv1 R205H mediates a robust resting-state H+ ‘shuttle’ conductance (GSH) at negative membrane potentials. Voltage-dependent GSH gating is measured at more negative voltages than the activation GAQ, indicating that VS activation is thermodynamically distinct from opening of the intrinsic H+ permeation pathway. A hallmark biophysical feature of Hv1 channels is a ~-40 mV/pH unit shift in the apparent voltage dependence of GAQ gating. We show here that changes pHO are sufficient to cause similar shifts in GSH gating, indicating that GAQ inherits its pH dependence from an early step in the Hv1 activation pathway. Furthermore, we show for the first time that Hv1 channels manifest a form of electromechanical coupling VS activation and GAQ pore opening. Second-site mutations of D185 markedly alter GAQ gating without affecting GSH gating, indicating that D185 is required for a late step in the activation pathway that controls opening of the aqueous H+ permeation pathway. In summary, this work demonstrates that the Hv1 activation pathway contains multiple transitions with distinct voltage and pH dependencies that have not been previously identified. The results reported here novel insight into the mechanism of VS activation in Hv1 and raise fundamental questions about the nature of pH-dependent gating and electromechanical coupling in related VS domain-containing ion channels and phosphatases.

Title: Molecular dynamics simulations of ion channel TRPA1 Author: Mgr. Vlastimil Zíma Institute: Institute of Physics of Charles University Supervisor: RNDr. Ivan Barvík, PhD., Institute of Physics of Charles Uni- versity Abstract: The ion channel TRPA1 is one of the members of the transient receptor potential channel family. These channels have recently been an im- portant objective of research, because they play important roles in various cellular processes and organismic mechanisms. Especially they are involved in most of the senses. We focused mainly on the TRPA1 ion channel due to its involvement in the pain sensation in humans. Because the molecular mechanisms behind the gating of this channel are not fully understood, their description is a key for a design of new analgesics targeting this channel. We used a homology modeling and molecular dynamics simulations in conjunc- tion with electrophysiological experiments to provide a valuable new insight into the channel mechanisms. We contributed by describing of a putative binding site for calcium ions. Further, many functionally important amino acids were found in the S1-S4 transmembrane domain. Keywords: voltage-gated ion channel, TRPA1 channel, molecular dynamics, homology modeling 1