A Novel Family of Insect-Selective Peptide Neurotoxins Targeting Insect Large-Conductance Calcium-Activated K⁺ Channels Isolated from the Venom of the Theraphosid Spider Eucratoscelus constrictus

Abstract

Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (-TRTXEc2a, -TRTX-Ec2b, and -TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that -TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K ϩ (BK Ca ) channel currents with IC 50 values of 3 to 25 nM. In addition, -TRTX-Ec2a caused the inhibition of insect delayed-rectifier K ϩ currents, but only at significantly higher concentrations.-TRTX-Ec2a and -TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous -TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, -TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, -TRTX-Ec2a and -TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BK Ca channels. This study therefore establishes these toxins as tools for studying the role of BK Ca channels in insects and lead compounds for the development of novel insecticides.

Conopeptide Vt3.1 Preferentially Inhibits BK Potassium Channels Containing β4 Subunits via Electrostatic Interactions

“…Previous studies have identified peptide toxins that modulate BK channels from venoms of various animals including scorpion (28 -30), cone snail (31), snake (32), bee (33), and spider (34). Among these toxins, charybdotoxin (ChTx) and iberiotoxin (IbTx) have been intensively studied for molecular pharmacology and used as an effective tool to identify BK channels in various tissues that contributed greatly in the understanding (Fig.…”

Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors

“…Thus: I BK(Ca) control = protocol 1 -protocol 3; while I BK(Ca) toxin = protocol 2 -protocol 3. 1 mM CdCl 2 and 5 mM 4-AP were included in the external solutions to block Ca V channel currents and I K(A) , respectively (Grolleau and Lapied, 1995;Gunning et al, 2008;Windley et al, 2011). Test pulses to +30 mV for 100 ms from a holding potential (V h ) -80 mV delivered at 0.1 Hz were used to evoke outward non-inactivating K DR and K Ca 1.1 channel currents.…”

“…Consequently, K Ca 1.1 channel current isolation was achieved using the following procedure (Gunning et al, 2008;Windley et al, 2011). Initially, both I K(DR) and I K(Ca) were recorded concurrently (protocol 1).…”

“…DUM neurons, isolated from the terminal abdominal ganglion (TAG) of unsexed American cockroaches (Periplaneta americana), were used for all patch-clamp experiments in this study (Gunning et al, 2008;Lapied et al, 1989;Windley et al, 2011). Single adult DUM neurons were isolated from cockroach TAG through mechanical and enzymatic dissociation, as previously described (Gunning et al, 2008;Windley et al, 2011).…”

“…Single adult DUM neurons were isolated from cockroach TAG through mechanical and enzymatic dissociation, as previously described (Gunning et al, 2008;Windley et al, 2011). Briefly, TAG were dissected from the ventral nerve cord and transferred to Ca 2+ /Mg 2+ -free NIS containing (in mM): NaCl 180, KCl 3.1, Nhydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) 10 and D-glucose 20.…”