Broadband Tuning the Voltage Dependence of a Sodium Channel

Thesis (Ph.D.)--University of Washington, 2019Mammalian voltage-gated sodium channels are comprised of four homologous domains, each containing a functionally distinct voltage-sensing domain (VSD). In contrast, the bacterial sodium channel NaVAb is a homotetramer, which enables investigation into the voltage-dependent gating mechanism without needing to account for heterogenic VSD contributions to the total signal. We endeavored to alter the properties of the VSD such that it moves in response to voltage changes over a wide range of voltages. By shifting the voltage dependence of activation in this manner, the channels can be used a means to better understand the structural features that control voltage sensing. We hypothesized that a VSD that activates only at voltages above 0 mV could be used as a tool to study the resting state with structural biology and further our understanding of the structural basis of voltage sensation. Human NaV1.7 has been identified as a drug target for the treatment of a multitude of pain disorders, thus we created several chimeric channels with human NaV1.7 DII residues comprising the extracellular portion of the VSD and NaVAb residues in the S0, S4-S5 linker, and pore domain. Channels with entirely human VSDs and ones wherein the human residues extended to the level of the intracellular negative cluster did not show measurable sodium current when transiently transfected into Sf9 cells with a GFP-P2A stoichiometric fluorescent indicator. Less humanized VSDs were functional and displayed a range of voltage-dependencies of activation V1/2 values and opening rates. Several single amino-acid substitutions whose effects were studied in other sodium and potassium channels further modified the voltage-dependence of gating in these chimeras. The combinatorial effect of these substitutions on activation was examined, and it was found that by combining mutations that produce strong effects on their own, drastic shifts in the voltage dependence of channel opening could be seen. In several cases, this did not translate to a substantial change in the voltage dependence of the gating charge movement itself, but it is possible that intermediate states were stabilized and impeded the fast movement of the voltage sensor. One combination of triple mutations, however, shifted channel opening and charge movement to more positive potentials, and this was used in combination with intersubunit disulfide crosslinking to stabilize a resting state of the voltage sensor in a cryo-EM structure at a resolution of 3.9 Ã . This structure showed an 11 Ã downward movement of the of the gating charges which is consistent with the sliding helix model of voltage sensor activation. The S4-S5 linker moves 9.6 Ã in the gating process as it constricts around the base of the pore and sterically prevents its opening. These findings enhance our understanding of the voltage sensation process and its translation to the opening of the pore of sodium channels