A single-nucleotide polymorphism (a guanine-to-adenine substitution) in the gene encoding the rat GABA_A receptor α6 subunit (Gabra6) is common in Sprague-Dawley rats obtained from Charles River Laboratories. Shown from left to right are the sequence chromatograms from a homozygous Gabra6^100R/100R, a heterozygous Gabra6^100R/100Q and a homozygous Gabra6^100Q/100Q rat. The lower panel shows the sequence of each Gabra6 genotype along with the corresponding amino acid translation (bottom). Gray boxes denote the codon for amino acid position 100.

Tonic GABA current in granule cells is enhanced by low concentrations of ethanol in Gabra6^100R/100R and Gabra6^100Q/100Q rats. (a,b) Tonic currents recorded from Gabra6^100R/100R (a) or Gabra6^100Q/100Q (b) granule cells showing that 10 mM ethanol enhances the tonic current in the continuous presence of 300 nM GABA, TTX, NBQX and NO-711. (c) Summary data indicating that in response to 10 mM ethanol, significant increases in tonic current are observed in Gabra6^100R/100R granule cells (P < 0.05, n = 4) and larger enhancements are found in Gabra6^100Q/100Q granule cells (P < 0.05, n = 4).

The α6-R100Q polymorphism leads to a marked increase in ethanol sensitivity when expressed with β3 and δ subunits. (a) GABARs of the indicated subunit compositions were expressed in X. laevis oocytes and activated by steady-state GABA (300 nM ~EC₃₀). Brief applications of ethanol at the indicated concentrations (in mM) enhance the current in a dose-dependent manner. (b) Dose-response curves for ethanol showing the peak enhancement of GABA current. Shown are wild-type and mutant versions of subunit combinations that are likely to be responsible for tonic GABA current in granule cells: α6β2δ (cross, n = 6), α6β3δ (asterisk, n = 10), α6(R100Q)β2δ (inverted triangles, n = 7) and α6(R100Q)β3δ (upright triangles, n = 8). Note that the β3 and δ subunits are required for the R100Q mutation to exert an effect and that replacement of β2 with β3 leads to an almost tenfold increase in ethanol sensitivity. Other combinations of subunits expressed in granule cells are summarized in Table 1.

Ethanol enhances granule cell tonic GABA current, and the enhancement is larger in Gabra6^100Q/100Q rats. (a,b) Tonic GABA currents in granule cells recorded from Gabra6^100R/100R (a) or Gabra6^100Q/100Q (b) slices in the presence of indicated concentrations of ethanol or in a saturating concentration (10μM) of the GABAR antagonist SR95531. To the right are histograms of all points in each segment. Gaussian functions have been fit to each condition and are superimposed. The dashed lines indicate the mean current from these fits. (c) Summary of the mean ± s.e.m. percentage change in tonic current amplitude caused by 30 or 100 mM ethanol in the two genotypes (wild type, n = 5; mutant, n = 7 granule cells). Ethanol caused a significantly larger enhancement of mutant versus wild-type currents. Mean tonic GABA current under control conditions was −9.7 ± 1.7 pA and did not differ significantly between the two genotypes (P > 0.4). (d) Ethanol-induced increases in sIPSC frequency are significantly larger in mutant granule cells (Gabra6^100R/100R, n = 5; Gabra6^100Q/100Q, n = 7 granule cells). No significant changes were observed in either the amplitude (Gabra6^100R/100R, 112.43 ± 12.57% of control, P > 0.49, n = 5; Gabra6^100Q/100Q, 101.55 ± 2.42% of control, P > 0.7, n = 7) or the decay of sIPSCs (Gabra6^100R/100R, 90 ± 12% of control, P > 0.42, n = 4; Gabra6^100Q/100Q, 96 ± 18% of control, P > 0.77, n = 6) in ethanol (data not shown).

Rats homozygous for the α6-100Q polymorphism show increased alcohol-induced motor impairment as compared with Gabra6^100R/100R rats. Motor coordination was assessed by testing Gabra6^100R/100R (open circles) and Gabra6^100Q/100Q (filled circles) rats in an accelerating rotarod test. (a–d) Performance was measured as the latency to fall from the rotating rod before (‘Pre’) and 20, 40 and 60 min after intraperitoneal injection of saline (a; n = 5 rats each group), 0.75 g kg⁻¹ (b, n = 7 and 8 rats for Gabra6^100R/100R and Gabra6^100Q/100Q, respectively), 1 g kg⁻¹ (c; n = 6 and 7 rats for Gabra6^100R/100R and Gabra6^100Q/100Q, respectively) or 1.25 g kg⁻¹ ethanol (d; n = 8 rats each group). Tests on the three postinjection data points yielded the following statistics: saline controls, Gabra6^100R/100R versus Gabra6^100Q/100Q: F_2,7 = 0.50, P = 0.63; 0.75 g kg⁻¹ ethanol, Gabra6^100R/100R versus Gabra6^100Q/100Q: F_2,12 = 6.66, P = 0.011; 1 g kg⁻¹ ethanol, Gabra6^100R/100R versus Gabra6^100Q/100Q: F_2,10 = 5.41, P = 0.026; 1.25 g kg⁻¹ ethanol, Gabra6^100R/100R versus Gabra6^100Q/100Q: F_2,13 = 135.6, P < 0.001. (e,f) Latency (e) and duration (f) of LORR was determined after intraperitoneal injection of 3 g kg⁻¹ ethanol (n = 9 for each group) and did not differ in Gabra6^100Q/100Q versus Gabra6^100R/100R rats (P = 0.51 for LORR latency, P = 0.81 for LORR duration).