Functional Expression of Drosophila para Sodium Channels Modulation by the Membrane Protein TipE and Toxin Pharmacology

The Drosophila para sodium channel a subunit was expressed in Xenopus oocytes alone and in combination with tipE , a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: ( a ) toxin II from Anemonia sulcata , which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity ( K d > 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; ( b ) Para sodium channels are . 10-fold more sensitive to block by tetrodotoxin; and ( c ) modification by the pyrethroid insecticide permethrin is . 100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels. key words: Na 1 channels • pyrethroid • insecticide • toxin • Xenopus oocyte i n t r o d u c t i o n Voltage-activated sodium channels are transmembrane proteins that provide the current pathway for rapid depolarization of muscles, nerves, and other electrically excitable cells (Hodgkin and Huxley, 1952; Catterall, 1992). Most of our current understanding about the structure and function of sodium channels has come from studying heterologously expressed mammalian sodium channels (Catterall, 1992). Although mammalian sodium channels contain as many as three subunits ( a , b 1 , and b 2 ), the ionic selectivity, time and voltage dependence of activation, modulation by protein kinases A and C, and most pharmacological properties are reconstituted by expression of the single core a subunit (Catterall, 1992). Coexpression of the a subunit with the b 1 subunit sometimes enhances expression and alters the time and voltage dependence of inactivation (Isom et al., 1992; Patton et al., 1994). While sodium channels have also been cloned from a variety of invertebrate organisms, little is known about the structure–function relationships of these sodium channel subunits because we and our collaborators (Feng et al., 1995 a ) have only recently identified conditions for heterologous expression of a representative invertebrate sodium channel from Drosophila. Drosophila provides a useful system to study sodium channels because it allows a combination of biophysical, molecular genetic, and transgenic techniques to facilitate both in vivo and heterologous expression studies. The gene encoding a sodium channel a subunit in Drosophila, paralytic (para) , has been cloned, and the entire cDNA sequence determined (Loughney et al., 1989; Ramaswami and Tanouye, 1989; Thackeray and Ganetzky, 1994). The predicted polypeptide is z 50% identical to vertebrate neuronal sodium channel a subunits and has four internally homologous domains like those conserved in all other voltage-gated sodium channels (Guy and Conti, 1990; Catterall, 1992). The para locus encodes the predominant class of voltage-activated sodium channels expressed in Drosophila neurons (Hong and Ganetzky, 1994), and the primary transcript from the para gene undergoes a developmentally regulated complex pattern of alternative splicing that potentially generates over 100 different sodium channel isoforms (Thackeray and Ganetzky, 1994; O’Dowd et al., 1995). Relatively little is known about the functional significance of these alternative exons, but the available evidence indicates that the splice variants could provide an important basis for structure-activity studies (O’Dowd et al., 1995 b ). Furthermore, sodium channels R.A.G. Reenan’s current address is Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030. Address correspondence to Dr. Charles Cohen, Merck Research Laboratories, P.O. Box 2000, Rm. 80N-31C, Rahway, NJ 07065. FAX: 908-594-3925; E-mail: [email protected] on M ay 5, 2017 D ow nladed fom Published August 1, 1997