Drosophila melanogaster GRD and LCCH3 subunits form heteromultimeric GABA-gated cation channels

Abstract

In addition to its action as a fast inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to mediate excitatory action by activating cation currents in some cell types in invertebrates. However, to date no GABA receptor capable of mediating such action has been identified at the molecular level in insects. Using a systematic expression screening approach, we found that the Drosophila ligand-gated ion channel subunits GRD and LCCH3 combine to form cation-selective GABA-gated ion channels when coexpressed in Xenopus laevis oocytes. The heteromultimeric receptor is activated by GABA (EC50=4.5 microm), muscimol (EC50=4.8 microm) and trans-4-aminocrotonic acid (EC50=104.5 microm), and partially by cis-4-aminocrotonic acid (EC50=106.3 microm). Picrotoxin effectively blocked the GABA-gated channel (IC50=0.25 microm), but bicuculline, TPMTA, dieldrin and lindane did not. The benzodiazepines flunitrazepam and diazepam did not potentiate the GABA-evoked current. Our data suggest that heteromultimeric channels composed of GRD and LCCH3 subunits form GABA-gated cation channels in insects.

Figure 1

(a) Oocytes were voltage clamped (V_h=−80 mV) 3–7 days after injection of 25 ng of various cRNA mixes. GABA applied at 1 mM for 10 s produced inward currents only in oocytes coexpressing GRD and LCCH3. The duration of application of agonists is indicated by bars. (b, c) Currents evoked by GABA and muscimol in oocytes co-injected with cRNA encoding GRD and LCCH3 subunits voltage clamped at –60 mV. (d) Agonist profile of oocytes coexpressing GRD and LCCH3. Recordings were performed as in (b, c). Each point represents the average value of four to seven oocytes. Data were normalized to the maximum GABA response (100 μM) seen in each oocyte. The concentration–response curve was fitted to the logistic equation.

Figure 2

(a) Antagonist profile of oocytes coexpressing GRD and LCCH3. Oocytes were voltage clamped (V_h=−80 mV) and 6 μM GABA was co-applied with various concentrations of PTX, dieldrin or bicuculline. (b) Concentration–block curve for PTX. Each point represents the average value of three to five oocytes. Concentration–response curves were fitted to the logistic equation.

Figure 3

(a) Current–voltage relationship. The averaged peak currents of the I/V curves were determined under varying external ionic conditions (normalized to the response in 100% Na⁺, 100% Cl⁻ Ringer): 100% Na⁺, 100% Cl⁻ Ringer (n=9); 25% Na⁺, 100% Cl⁻ Ringer (n=4); 100% Na⁺, 25% Cl⁻ Ringer (n=4). (b) Reversal potential shifts for ionic substitution experiments. Reversal potentials were measured in solutions with varying amounts of sodium (n=4) or chloride ions (n=4). (c) The pore-forming M2 domain of GRD (CAA55144) contains the molecular determinants for cation selectivity and differs in the three critical positions −2′, −1′ and 13′ (Wotring et al., 2003) from the ligand-gated chloride channel RDL (AAA28556) and the rat ρ1-GABA receptor (P50572) and resembles ligand-gated cation channels such as EXP-1 (AY383563), the rat 5HT₃ receptor (NP_038589) and the rat nicotinic α7 ACh receptor (NP_036964) at these positions. AFF48539 and LCCH3 (AAB27090) show these differences to a lesser extent.