Two γ2L subunit domains confer low Zn2+ sensitivity to ternary GABAA receptors

The sensitivity of GABAA receptors (GABARs) to Zn2+ inhibition depends on subunit composition. The predominant neuronal forms of mammalian GABARs, αβγ and αβδ, are differentially sensitive to Zn2+ inhibition; αβγ receptors are substantially less sensitive than αβδ receptors. Recently, functional domains involved in Zn2+ sensitivity have been identified in α and β subunits. Our aim in the present study was to localize functional domains of low Zn2+ sensitivity within γ2L subunits. Chimeric subunits were constructed by progressively replacing the rat γ2L subunit sequence with that of the rat δ subunit sequence. Whole-cell currents were recorded from mouse L929 fibroblasts coexpressing wild-type rat α1 and β3 subunits with a chimeric δ-γ2L subunit. Unlike α and β subunits, the γ2L subunit was found to contain a determinant of low Zn2+ sensitivity in the N-terminal extracellular region. In addition, we identified determinants in the M2 segment and the M2-M3 loop of the γ2L subunit that are homologous to those found in β and α subunits. We postulate that the interface between the latter two domains, which may form the outer vestibule of the channel, represents a single functional domain modulating Zn2+ sensitivity. Thus, the Zn2+ sensitivity of ternary GABARs appears to be determined by two functional domains, one in the N-terminal extracellular region and one near the outer mouth of the channel. The γ-aminobutyric acid type A receptor (GABAR) is the major inhibitory neurotransmitter receptor in the mammalian central nervous system. Functional mammalian GABARs are thought to be pentameric combinations of homologous subunits drawn from seven different families with multiple subtypes: α(1-6), β(1-3), γ(1-3), δ, e, π and θ (Macdonald & Olsen, 1994; Davies et al. 1997; Hedblom & Kirkness, 1997; Bonnert et al. 1999). Like other members of the ligand-gated ion channel gene superfamily, GABAR subunits have a putative membrane topology consisting of a large N-terminus, four membrane-spanning segments (M1-M4), one extracellular and two intracellular loops connecting the membrane-spanning segments (M1-M2, M2-M3, M3-M4), and an extracellular C-terminus. It has been suggested that neuronal GABARs are predominantly ternary αβγ and αβδ combinations (Angelotti et al. 1993; McKernan & Whiting, 1996). The divalent cation Zn2+ has been proposed to be an endogenous modulator of synaptic transmission (Smart et al. 1994; Harrison & Gibbons, 1994). Its ability to inhibit native GABARs, however, varies with neuronal type, age and activity (Smart et al. 1994; Kapur & Macdonald, 1997). Studies of recombinant receptors indicate that the inhibitory activity of Zn2+ on GABAR currents depends on subunit composition (Draguhn et al. 1990; Smart et al. 1991). Binary αβ GABARs have a high sensitivity to Zn2+ with IC50 values ranging from 0.1 to 1 μm (Draguhn et al. 1990; Smart et al. 1991; Wooltorton et al. 1997; Horenstein & Akabas, 1998). In ternary αβX GABARs (where X is δ, γ, e or π), Zn2+ sensitivity is decreased. Receptors containing π subunits have a Zn2+ IC50 of ≈2 μm (Neelands & Macdonald, 1999), receptors containing δ subunits have Zn2+ IC50 values ranging from 5 to 16 μm (Saxena & Macdonald, 1996; Krishek et al. 1998), and receptors containing e subunits have a Zn2+ IC50 of ≈40 μm (Whiting et al. 1997; Neelands et al. 1999). Incorporation of a γ subunit, however, has an even greater effect on GABAR Zn2+ sensitivity, resulting in IC50 values that range from 20 to 600 μm (Saxena & Macdonald, 1996; Burgard et al. 1996; Krishek et al. 1998). Functional domains involved in Zn2+ sensitivity have been identified in two GABAR subunit families, α and β. The extracellular end of the M2 segment in the β subunit has been shown to be a major determinant of Zn2+ sensitivity in β homomers and αβ receptors (Wooltorton et al. 1997; Horenstein & Akabas, 1998). The M2-M3 extracellular loop of the α6 subtype has been shown to confer higher Zn2+ sensitivity to α6β3γ2L GABAR isoforms as compared to α1β3γ2L receptors (Fisher & Macdonald, 1998). In addition, in GABAC receptors, which are structurally homologous to GABARs, the N-terminus has an essential role in conferring Zn2+ sensitivity to ρ1 homomers (Wang et al. 1995). Such functional domains, however, have yet to be identified for other GABAR subunit families including δ and γ. To determine which domains of the γ2L subunit subtype are involved in conferring low Zn2+ sensitivity to GABARs, we constructed GABAR chimeras by replacing varying lengths of the rat γ2L subunit with the homologous sequence of the rat δ subunit. These chimeric subunits were coexpressed in mouse L929 cells with wild-type α1 and β3 subunits, and whole-cell currents were elicited by application of GABA. The relative Zn2+ sensitivities of GABARs containing the chimeric subunits revealed that two different and non-contiguous γ2L subunit domains, one in the N-terminal extracellular region and one near the outer mouth of the channel, contributed to the regulation of Zn2+ sensitivity.