Biophysical and pharmacological characterization of insect voltage-gated sodium channels

Voltage-gated sodium channels are membrane proteins that play critical roles in generation and propagation of action potentials. In this dissertation, I described the biophysical and pharmacological properties of the insect voltage-gated sodium channel in primary cultured central neurons as well as in a heterologous expression system, Xenopus oocytes. Using the techniques of site-directed mutagenesis, patch clamp and two-electrode voltage-clamp conducted an investigation of the molecular mechanisms underlining site-insensitive pyrethroid knockdown resistance (kdr).; In cultured central neurons, sodium channels from kdr housefly, which carried L1014F mutation in Para-like sodium channel, show 5.4 mV and 6.1 mV positive shifts of voltage-dependent activation and steady-state inactivation, respectively. The kdr sodium channels show 42-fold lower sensitivity to permethrin at 5% channel modification.; In Xenopus oocytes expressing Para channels with the mutations V421M and L1029H identified from H. virescens study, I demonstrated that both mutations confer channel insensitivity to permethrin, with the L1029H having a more pronounced effect. Both mutations also alter the voltage-dependent gating properties of the channel, but L1029H less so than V421M. These results suggest that V421M exacts a higher fitness cost than L1029H. These studies demonstrate that single point mutations in the sodium channel account for the kdr mechanism.; Both kdr mutations abolish the channel sensitivity to aconitine, but not to batrachotoxin and veratridine in the oocyte expression study. The mutation V421K completely blocks the channel sensitivity to all of the 3 toxins. These results indicate that sodium channel site 2 toxins have similar or over-lapping receptors.; I also demonstrated that permethrin dissociates from the sodium channel according to two off-rates, fast and slow. Aconitine accelerates the slow off-rate, suggesting an allosteric modulation of the permethrin binding site. The two different off-rates suggest that pyrethroids have at least two binding sites on sodium channels.; The complete coding sequence of a putative sodium channel in Drosophila (dscl) is described. Dscl has 2851 amino acids with a calculated molecular weight 322.1 kDa. Phylogenetic analysis of 17 sodium channels showed that Para channel is grouped with vertebrate sodium channels; DSCl is grouped with jellyfish putative sodium channels.