Sodium channels in central neurons of the tobacco budworm, Heliothis virescens: basic properties and modification by scorpion toxins

Abstract

Voltage-activated sodium channels in central neurons of larval and adult Heliothis virescens were characterized using whole-cell patch clamp techniques. Macroscopic currents showing rapid activation and inactivation kinetics were uniformly sensitive to tetrodotoxin (IC₅₀=1.9 nM). Currents began to activate at voltage steps to −45 mV and reached half maximal at −30 mV. Fast inactivation was evident at voltages as negative as −75 mV and reached half maximal at −50 mV. Full recovery from inactivation occurred within 1 to 2 ms. Currents in larval neurons exhibited similar properties to those of adult neurons, except for the rate of fast inactivation (t₁), which was significantly slower in larval neurons. The biophysical properties of sodium channels remained unchanged for up to 3 days in culture. Two insecticidal neurotoxins, LqhαIT and AaIT, produced distinctly different modifications of H. virescens sodium channels. LqhαIT slowed channel inactivation, while AaIT specifically shifted voltage-dependent activation to more negative potentials. Overall, the results indicate that sodium channels in H. virescens neurons exhibit biophysical characteristics similar to those of vertebrates, yet possess pharmacological uniqueness with respect to scorpion toxin modification.

Introduction

Electrical and chemical signaling in the nervous system is of fundamental importance to the integration of external and internal stimuli. Proper signaling depends on a finely tuned series of ion-channel-mediated events mediating electrical activity and transduction at synapses. Slight changes in ion channel properties can lead to drastic changes in normal physiological functions. It is therefore understandable that many natural toxins and insecticides target ion channels in the nervous system. Arthropod venoms in particular contain a rich array of toxins which, through disruption of channel function, serve as effective biochemical weapons in prey capture or defense.

A major target of arthropod toxins is the voltage-activated sodium channel, which plays a critical role both in action potential propagation as well as transduction of electrical current to transmitter release from the nerve terminal. All sodium channels examined to date contain a large glycoprotein α-subunit of 230–280 kDa associated with one or two smaller auxiliary subunits (Loughney et al., 1989; Catterall et al., 1992; Kallen et al., 1993; Feng et al., 1995). The α-subunit forms the functional pore and contains molecular machines for channel opening, inactivation and closing. At least six non-overlapping binding sites have been identified for neurotoxins that act either as pore blockers or gating modifiers (Catterall, 1988; Catterall et al., 1992; Hayashi and Gard, 1994; Cestele et al., 1995). A few accounts of sodium channel properties in insect neurons have appeared, and recently the para sodium channel of Drosophila was successfully cloned and expressed (Feng et al., 1995; Warmke et al., 1997). However, as yet no detailed characterization of lepidopteran neuronal sodium channels has been published.

Insect sodium channels are targeted by a growing list of arthropod venom toxins which modify their properties, leading to either excitatory or depressant symptomologies and consequent paralysis (Adams et al., 1989; Zlotkin et al., 1994; Krapcho et al., 1995). In particular, scorpion venoms contain small, single-chain polypeptide toxins with high toxicity to animals (Catterall et al., 1992; Martin-Eauclaire and Courad, 1995). The majority of scorpion neurotoxins specifically targeting sodium channels can be classified into three groups: (1) α-scorpion toxins, which bind to Site 3 on sodium channels and slow inactivation; (2) β-scorpion toxins, which bind to Site 4 and shift the voltage dependence of sodium channel activation to more negative potentials; and (3) a third site specific to insect sodium channels leading to either excitatory or depressant symptomologies (Zlotkin et al., 1994).

With their selective toxicity for insects over mammals, insecticidal scorpion toxins have become useful pharmacological tools for the study of insect sodium channel uniqueness and for the design of selective bioinsecticides. Scorpion toxin genes have been incorporated into insect pathogens resulting in enhanced speed of kill over wild-type viruses (Stewart et al., 1991; Tomalski and Miller, 1991; Bonning and Hammock, 1996). This strategy provides an excellent mechanism for delivering hydrophilic polypeptide toxins to the pest organism. For example, a baculovirus expressing the AaIT gene reduced time to kill of Heliothis virescens larvae by about 30 to 40%, and dramatically reduced feeding (McCutchen and Hammock, 1994). Since considerable progress has been made in incorporating genes for some of these toxins into engineered baculoviruses, it is of interest to fully characterize the actions and selectivities of these toxins on sodium channels of pest insects.

Most major agricultural crop pests belong to the Order Lepidoptera. Among the most serious pests is the tobacco budworm, Heliothis virescens, which causes significant economic damage to cotton and tobacco in North America. Here we characterize native H. virescens sodium channels in central neurons and their modification by two distinct excitatory insecticidal scorpion toxins, LqhαIT and AaIT.