Price KL and Lummis SC (2014), An atypical residue in the pore of Varroa destr...

XB-ART-54038

Insect Biochem Mol Biol 2014 Dec 01;55:19-25. doi: 10.1016/j.ibmb.2014.10.002.

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An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation.

Price KL , Lummis SC .

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GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6'M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6'M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6' residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.

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Species referenced: Xenopus laevis
Genes referenced: gabarap

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	Fig. 1. A. Model of the RDL receptor transmembrane region showing two opposing subunits lining the pore. The residues investigated in this study are depicted. B. Exon organisation of the rdl gene denoting the position of the primers used for amplification of the Varroa M2 region. C. Sequence of the Varroa RDL receptor (V. De) amplified by these primers and its alignment with corresponding Drosophila (D. me) and Apis (A. me) RDL sequences.
	Fig. 2. A. Representative current traces from electrophysiological measurements of GABA responses from WT and mutant RDL receptors. GABA was applied for 40 s (0.1–3 μM GABA) or 15 s (10–100 μM GABA). B. Concentration-response curves for activation of RDL receptors by GABA. Parameters obtained from these curves are shown in Table 1.
	Fig. 3. Concentration-inhibition curves for inhibition of GABA responses by picrotoxin. Parameters obtained from these curves are shown in Table 2.
	Fig. 4. A: Example traces of WT and T6′M RDL receptors activated by 1 μM GABA (black line) in the absence and presence (grey line) of 100 μM thymol. B: GABA concentration–response curves in the absence (open circles) and presence (filled circles) of 100 μM thymol. % Imax values are WT:94 ± 3; N-7′H:82 ± 4, T6′M:35 ± 9; 4M2:52 ± 4; A20′S:81 ± 6; and A21’Q:89 ± 4; * = significantly different to WT, p < 0.05. Other parameters obtained from these curves are shown in Table 3.