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Two 2L subunit domains confer low Zn²⁺ sensitivity to ternary GABA_A receptors

Naomi Nagaya * and Robert L. Macdonald *†

	ABSTRACT

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1. The sensitivity of GABA_A receptors (GABARs) to Zn²⁺ inhibition depends on subunit composition. The predominant neuronal forms of mammalian GABARs, and , are differentially sensitive to Zn²⁺ inhibition; receptors are substantially less sensitive than receptors. Recently, functional domains involved in Zn²⁺ sensitivity have been identified in and subunits. Our aim in the present study was to localize functional domains of low Zn²⁺ sensitivity within 2L subunits.

2. Chimeric subunits were constructed by progressively replacing the rat 2L subunit sequence with that of the rat subunit sequence. Whole-cell currents were recorded from mouse L929 fibroblasts coexpressing wild-type rat 1 and 3 subunits with a chimeric -2L subunit.

3. Unlike and subunits, the 2L 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 2L subunit that are homologous to those found in and subunits.

4. 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.

	INTRODUCTION

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The -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: (1-6), (1-3), (1-3), , , and (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 and 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 GABARs have a high sensitivity to Zn²⁺ with IC₅₀ values ranging from 0.1 to 1 M (Draguhn et al. 1990; Smart et al. 1991; Wooltorton et al. 1997; Horenstein & Akabas, 1998). In ternary X GABARs (where X is , , or ), Zn²⁺ sensitivity is decreased. Receptors containing subunits have a Zn²⁺ IC₅₀ of ~2 M (Neelands & Macdonald, 1999), receptors containing subunits have Zn²⁺ IC₅₀ values ranging from 5 to 16 M (Saxena & Macdonald, 1996; Krishek et al. 1998), and receptors containing subunits have a Zn²⁺ IC₅₀ of ~40 M (Whiting et al. 1997; Neelands et al. 1999). Incorporation of a subunit, however, has an even greater effect on GABAR Zn²⁺ sensitivity, resulting in IC₅₀ values that range from 20 to 600 M (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, and . The extracellular end of the M2 segment in the subunit has been shown to be a major determinant of Zn²⁺ sensitivity in homomers and receptors (Wooltorton et al. 1997; Horenstein & Akabas, 1998). The M2-M3 extracellular loop of the 6 subtype has been shown to confer higher Zn²⁺ sensitivity to 632L GABAR isoforms as compared to 132L receptors (Fisher & Macdonald, 1998). In addition, in GABA_C receptors, which are structurally homologous to GABARs, the N-terminus has an essential role in conferring Zn²⁺ sensitivity to 1 homomers (Wang et al. 1995). Such functional domains, however, have yet to be identified for other GABAR subunit families including and .

To determine which domains of the 2L subunit subtype are involved in conferring low Zn²⁺ sensitivity to GABARs, we constructed GABAR chimeras by replacing varying lengths of the rat 2L subunit with the homologous sequence of the rat subunit. These chimeric subunits were coexpressed in mouse L929 cells with wild-type 1 and 3 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 2L 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.

	METHODS

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Construction of expression vectors and mutagenesis

Full-length cDNAs for the rat GABAR 1 (Dr A. Tobin, University of California, Los Angeles, CA, USA), 3 (Dr D. Pritchett, University of Pennsylvania, Philadelphia, PA, USA), (Dr K. Angelides, Baylor College of Medicine, Houston, TX, USA) and 2L subunits (F. Tan, University of Michigan) were subcloned into the expression vector pCMVneo (Huggenvik et al. 1991). Chimeras were constructed using the splice overhang extension method. Point mutations were generated using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA). Oligonucleotide primers were synthesized by the University of Michigan DNA synthesis core. Sequences of chimeras and point mutants were verified by fluorescent DNA sequencing (University of Michigan DNA sequencing core).

Cell culture and transient transfection

The mouse fibroblast cell line L929 (American Tissue Type Collection, Rockville, MD, USA) was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % horse serum, 100 i.u. ml^-1 penicillin and 100 g ml^-1 streptomycin (all from Gibco-BRL, Grand Island, NY, USA). Cells were maintained in a 37 °C incubator with 95 % air-5 % CO₂ in 10 cm culture dishes. Cells were passaged every 3-4 days using 0.5 % trypsin-0.2 % EDTA (Boehringer-Mannheim, Indianapolis, IN, USA) in divalent-free, phosphate-buffered saline (PBS; 10 mM Na₂HPO₄, 0.15 mM NaCl, pH 7.3). Twenty-four hours prior to transfection, cells were seeded at a density of 300 000 in 60 mm culture dishes. Cells were transfected using a modified calcium phosphate precipitation method. Plasmids encoding GABAR subunits were mixed in a 1:1:1 ratio using 3-4 g of each along with 2 g of pHook-1 (Invitrogen, San Diego, CA, USA), which encodes the cell surface antibody sFv. Following addition of DNA, cells were incubated for 4-5 h in 3 % CO₂ and shocked for 30 s with 15 % glycerol in BBS buffer (50 mM Bes, 280 mM NaCl, 1.5 mM Na₂HPO₄). The following day, transfected cells were selected and concentrated using Capture-Tec beads (magnetic beads coated with hapten; Invitrogen). Selected cells were replated on 35 mm culture dishes and used for recording about 24 h later.

Electrophysiological recording solutions and techniques

For whole-cell recording, the external bath solution consisted of (mM): 142 NaCl, 8.1 KCl, 6 MgCl₂, 1 CaCl₂, 10 glucose and 10 Hepes at pH 7.4 and osmolality between 311 and 325 mosmol kg^-1. The concentration of Mg²⁺ in the bath solution did not change the GABAR IC₅₀ values for Zn²⁺. The IC₅₀ values for the wild-type receptors 13 and 132L were similar to those reported previously in the literature (Gingrich & Burkat, 1998; Krishek et al. 1998). Recording electrodes were filled with an internal solution of (mM): 153 KCl, 1 MgCl₂, 5 K-EGTA, 10 Hepes and 2 Mg-ATP at pH 7.3 and osmolality adjusted to 295-300 mosmol kg^-1. These solutions provided a chloride equilibrium potential near 0 mV. Patch pipettes were pulled from microhaematocrit tubes made of soda-lime glass (i.d. = 1.1-1.2 mm, o.d. = 1.3-1.4 mm; Fisher Scientific, Pittsburgh, PA, USA) on a P-87 Flaming Brown puller (Sutter Instrument Co., San Rafael, CA, USA). Pipettes had resistances of 5-10 M and were coated with polystyrene Q-Dope (GC Electronics, Rockfield, IL, USA) before use. Currents were recorded with an Axoclamp 200A patch clamp amplifier (Axon Instruments, Foster City, CA, USA), DigiData 1200 interface (Axon Instruments) and Zenith Pentium computer as well as on Beta videotape. Series resistance was compensated by 90 %. GABA and ZnCl₂ were prepared as stock solutions of 100 mM in water. All working solutions were prepared on the day of the experiment by diluting stock solutions in external solution. Drugs were applied to cells using a modified U-tube delivery system with a 10-90 % rise time of 70-150 ms (Greenfield & Macdonald, 1996). GABA-induced currents were recorded at a holding potential of -75 mV. All experiments were performed at room temperature.

Analysis of whole-cell currents

Whole-cell currents were analysed off-line using Axoscope (Axon Instruments) and Prism software (GraphPad, San Diego, CA, USA). Normalized concentration-response data for the different isoforms were fitted with a four-parameter logistic equation:

I = I_max/(1 + 10^{(logEC50 - logX)nH}),

where X is the concentration of drug, n_H represents the Hill coefficient and I represents current expressed as a percentage of the maximum current elicited by saturating concentrations of GABA for each cell or in the case of Zn²⁺, as a percentage of the current elicited by GABA alone (I_max). Data in the figures were derived from a single fit of averaged responses from all cells. Data in Tables 1 and 2 were derived from fits of individual cells and are reported as means ± S.E.M. Statistical tests were performed by one-way ANOVA and Newman-Keuls multiple comparisons tests where P < 0.05. Results of significance tests are presented in the relevant table legends.

Expression of -2L chimeric GABARs

Although the and 2L subunit families share only 35 % homology with each other (Shivers et al. 1989), the four chimeras we constructed produced functional channels when coexpressed with wild-type and subunits. The GABA EC₅₀ values for all of the chimeric isoforms were distinct from those observed for binary receptors (~1 M; Angelotti et al. 1993), indicating that the chimeric subunits were incorporated and influenced channel properties.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Design of -2L chimeric subunits

To determine which domains of the 2L subunit were involved in conferring low Zn²⁺ sensitivity to ternary GABARs, four chimeric subunits were created by progressively replacing the wild-type 2L subunit sequence from the N-terminus with wild-type sequence. Figure 1 depicts the putative membrane topology for each of the -2L chimeras including the large N-terminus, the four transmembrane segments (M1-M4) and their interconnecting loops, and the extracellular C-terminus. These chimeras divided the GABAR subunit sequence into five sections: (1) the N-terminus, (2) the M1 segment, (3) the M1-M2 loop and the M2 segment, (4) the M2-M3 loop and (5) the M3 and M4 segments, the M3-M4 loop and the C-terminus.

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Figure 1. Schematic representation of four -2L chimeric subunits The putative membrane topologies for four -2L chimeras are shown. Above each subunit, the chimera name is given, and below each subunit, the splice sites are given by amino acid and residue number. For each subunit, the extracellular N- and C-termini are indicated (N, C) and the four putative transmembrane segments labelled (M1-M4). The extent of rat subunit sequence is represented by dashed lines whereas the extent of rat 2L subunit sequence is represented by continuous lines.

GABA sensitivity of GABARs containing wild-type and chimeric subunits

Wild-type and chimeric forms of the and 2L subunits produced functional GABAR channels when coexpressed with wild-type 1 and 3 subunits in L929 fibroblasts. Cells were voltage clamped at -75 mV, and whole-cell currents were elicited by application of GABA. Comparison of concentration-response curves for GABARs containing wild-type subunits indicated that 132L receptors had a higher EC₅₀ for GABA than 13 receptors (Fisher & Macdonald, 1997; P < 0.001; Table 1).

Currents from GABARs containing chimeric subunits were differentially sensitive to GABA (Fig. 2A). Two of the isoforms containing chimeric -2L subunits, 13-2L(M3e) and 13-2L(M1e), had GABA EC₅₀ values similar to that of GABARs containing the wild-type subunit (Fig. 2B, Table 1). The other two chimeric receptors, 13-2L(M2e) and 13-2L(M1i), had higher GABA EC₅₀ values that were similar to that of GABARs containing the wild-type 2L subunit (Fig. 2B, Table 1). These results suggest that the juxtaposition of the subunit transmembrane and/or loop sequences with the subunit N-terminal extracellular sequence could influence GABA binding and/or channel gating such that a higher GABA EC₅₀ value was observed.

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Figure 2. GABA sensitivity of GABARs containing chimeric -2L subunits A, representative whole-cell currents from L929 fibroblasts expressing 13-2L(M1e) receptors (upper left), 13-2L(M1i) receptors (upper right), 13-2L(M2e) receptors (lower left), or 13-2L(M3e) receptors (lower right). The indicated concentrations of GABA were applied for 6-12 s (horizontal bars) to cells voltage clamped at -75 mV. B, concentration-response curves for cells expressing 13-2L(M1e) receptors (), 13-2L(M1i) receptors (), 13-2L(M2e) receptors () and 13-2L(M3e) receptors () are shown by continuous lines. Concentration-response curves for wild-type 13 (left) and 132L (right) receptors are shown for comparison (dashed lines). The peak response to each concentration of GABA was normalized as a percentage of the maximum current response for each cell. Values are means ± S.E.M. Data for each isoform were fitted with a four-parameter logistic equation with the indicated EC50 values and Hill slopes (nH).

Zn²⁺ sensitivity of GABARs containing wild-type and chimeric subunits

Currents evoked from wild-type 13 and 132L receptors by EC₅₀ concentrations of GABA were differentially inhibited by coapplication of 10 M Zn²⁺ (Fig. 3A). The Zn²⁺ IC₅₀ of 132L receptors was significantly higher than that of 13 receptors (Fig. 3B, P < 0.001; Table 2).

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Figure 3. Zn2+ sensitivity of GABARs containing wild-type or 2L subunits A, representative whole-cell currents from L929 fibroblasts expressing 13 receptors (left) or 132L receptors (right). GABA or GABA plus 10 M Zn2+ was applied for 7 s (horizontal bars) to cells voltage clamped at -75 mV. The concentration of GABA used was near the EC50 value for the given isoform. B, concentration-response curves for cells expressing 13 receptors () or 132L receptors (). The peak response to each concentration of Zn2+ was normalized as a percentage of the maximum current response to GABA alone for each cell. Values are means ± S.E.M. Data for each isoform were fitted with a four-parameter logistic equation with the indicated IC50 values and Hill slopes (nH).

Currents evoked from chimeric GABARs by EC₅₀ concentrations of GABA were also differentially inhibited by coapplication of 10 M Zn²⁺ (Fig. 4A). Progressive replacement of the 2L subunit from the N-terminus with the subunit sequence produced a progressive decrease in Zn²⁺ IC₅₀ (Fig. 4B). The 13-2L(M1e) isoform had an average Zn²⁺ IC₅₀ that was substantially lower than that of 132L receptors (P < 0.001; Table 2). Replacement of the wild-type 2L subunit with this chimeric subunit accounted for 63 % of the log difference between Zn²⁺ IC₅₀ values of 132L and 13-2L(M3e) receptors, suggesting that the N-terminal extracellular domain contained a critical determinant. Replacement of additional 2L subunit sequence (the M1 segment) with the subunit sequence produced little change in Zn²⁺ sensitivity (Fig. 4B); the 13-2L(M1i) isoform had an average Zn²⁺ IC₅₀ that was unchanged from that of the 13-2L(M1e) isoform (Table 2), suggesting that the M1 segment was not involved in conferring low Zn²⁺ sensitivity. Extending the subunit sequence through the 2L subunit M1-M2 loop and the M2 segment, however, again decreased Zn²⁺ IC₅₀ (Fig. 4B). The 13-2L(M2e) isoform had an average Zn²⁺ IC₅₀ that was substantially lower than that of the 13-2L(M1e) isoform (Table 2). This chimeric subunit accounted for an additional 19 % of the log difference between Zn²⁺ IC₅₀ values of 132L and 13-2L(M3e) receptors, suggesting that the M1-M2 loop and M2 segment contained another determinant of low Zn²⁺ sensitivity. Extending the subunit sequence through the 2L subunit M2-M3 loop also decreased Zn²⁺ IC₅₀ (Fig. 4B). The 13-2L(M3e) isoform had an average Zn²⁺ IC₅₀ that was lower than that of the 13-2L(M2e) isoform (Table 2). This chimeric subunit accounted for an additional 18 % of the log difference between Zn²⁺ IC₅₀ values of the 132L and 13-2L(M3e) receptors, suggesting that the M2-M3 loop contained a third determinant of low Zn²⁺ sensitivity.

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Figure 4. Zn2+ sensitivity of GABARs containing chimeric -2L subunits A, representative whole-cell currents from L929 fibroblasts expressing 13-2L(M1e) receptors (upper left), 13-2L(M1i) receptors (upper right), 13-2L(M2e) receptors (lower left), or 13-2L(M3e) receptors (lower right). GABA or GABA plus 10 M Zn2+ was applied for 6-12 s (horizontal bars) to cells voltage clamped at -75 mV. B, concentration-response curves for cells expressing 13-2L(M1e) receptors (), 13-2L(M1i) receptors (), 13-2L(M2e) receptors (), and 13-2L(M3e) receptors () are shown by continuous lines. Concentration-response curves for wild-type 13 (left) and 132L (right) receptors are shown for comparison (dashed lines). The peak response to each concentration of Zn2+ was normalized as a percentage of the maximum current response to GABA alone for each cell. Values are means ± S.E.M. Data for each isoform were fitted with a four-parameter logistic equation with the indicated IC50 values and Hill slopes (nH).

Point mutations in the outer vestibule

Of the three structural determinants identified by the -2L chimeras, we proceeded to further delineate two adjacent domains: (1) the M1-M2 loop and the M2 segment and (2) the M2-M3 loop. We chose to focus on these adjacent regions since previous studies of GABARs had identified functional domains for Zn²⁺ in the M2 segment of subunits and in the M2-M3 loop of subunits, suggesting the presence of homologous domains in 2L and subunits (Wooltorton et al. 1997; Fisher & Macdonald, 1998; Horenstein & Akabas, 1998). We targeted the extracellular end of the M2 segment and the proximal end of the M2-M3 extracellular loop, subunit regions putatively associated with the outer vestibule of the channel. Specific amino acid residues in the wild-type and 2L subunits were targeted for site-directed mutagenesis based on studies of Zn²⁺ sensitivity involving other GABAR subunits.

The M2 segment. The putative channel-lining M2 segment is highly conserved across all GABAR subunits. There are, however, a few sequence differences among subunit families. Between the and 2L subunits, there are differences at four positions in the M2 sequence (Fig. 5A). Three differences occur in a triplet of amino acids at the extracellular end of the M2 segment. Residues in the 1 subunit that are homologous to the second and third position of the triplet, I270 and S271, have been shown to be water accessible in receptors (Xu & Akabas, 1996). Residues in the 1 and 3 subunits homologous to the third position of the triplet, H292 and H267, respectively, were shown to be major determinants of Zn²⁺ inhibition in homomers and binary receptors and also appeared to be water accessible (Wooltorton et al. 1997; Horenstein & Akabas, 1998). Therefore, we focused on the extracellular triplet of residues and made the mutations M278S, V279T, S280I (MVS STI) and S280M, T281V, I282S (STI MVS) in the and 2L subunits, respectively.

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Figure 5. Zn2+ sensitivity of GABARs containing M2 mutant subunits A, M2 segment sequences for the rat 1, 3, 2L and subunits are shown. Residue numbers are based on the mature protein amino acid sequences. Residues in the 1 and 3 subunits previously shown to be water accessible are underlined (Xu & Akabas, 1996; Horenstein & Akabas, 1998). The four sequence differences between the M2 segments of the 2L and subunits are in bold and italicized. The triplets at the extracellular ends of the 2L and M2 segments that were targeted for mutagenesis are underlined. B, representative whole-cell currents from L929 fibroblasts expressing 13(MVS STI) receptors (left) and 132L(STI MVS) receptors (right). GABA or GABA plus 10 M Zn2+ was applied for 7 s (horizontal bars) to cells voltage clamped at -75 mV. C, concentration-response curves for cells expressing 13(MVS STI) receptors () and 132L(STI MVS) receptors (). Concentration-response curves for wild-type 13 (left) and 132L (right) receptors are shown for comparison (dashed lines). The peak response to each concentration of Zn2+ was normalized as a percentage of the maximum current response to GABA alone for each cell. Values are means ± S.E.M. Data for each isoform were fitted with a four-parameter logistic equation with the indicated IC50 values and Hill slopes (nH).

Coexpression of or 2L M2 mutant subunits with wild-type 1 and 3 subunits resulted in functional GABARs. The GABA EC₅₀ values of the 13(MVS STI) and 132L(STI MVS) isoforms were similar to those of their respective wild-type receptors (Table 1), suggesting that the triple mutations in the M2 segment did not affect GABA sensitivity.

The Zn²⁺ IC₅₀ values of GABARs containing either of the M2 mutants differed from those of GABARs containing wild-type subunits (Fig. 5B and C). The 13(MVS STI) isoform had an average Zn²⁺ IC₅₀ that was substantially higher than that of the 13 isoform (Table 2). This mutant subunit accounted for 17 % of the log difference between Zn²⁺ IC₅₀ values of 132L and 13 receptors. The 132L(STI MVS) isoform had an average Zn²⁺ IC₅₀ that was substantially lower than that of the 132L isoform (P < 0.001; Table 2). This mutant subunit accounted for 48 % of the log difference between Zn²⁺ IC₅₀ values of 132L and 13 receptors. These results indicated that the extracellular end of the M2 segment plays an important role in determining the Zn²⁺ sensitivity of ternary GABARs. Introduction of the 2L triplet into the subunit background decreased Zn²⁺ sensitivity whereas removal of the triplet from the 2L subunit background increased Zn²⁺ sensitivity. The effects of these mutations, however, were not equivalent for the 2L and subunits (see Discussion).

To determine whether the influence of the M2 triplet on Zn²⁺ sensitivity was dependent on only one of the residues, we made a set of point mutations. Mutagenesis at the third position of the triplet in and subunits has been shown to influence the Zn²⁺ sensitivity of binary receptors (Wooltorton et al. 1997; Horenstein & Akabas, 1998). Therefore, point mutations were made at this position in the and 2L subunits at S280 and I282, respectively. In addition, the contributions of the first and second residues of the triplet were tested by making the point mutations M278S and V279T in the subunit.

Coexpression of the or 2L M2 triplet mutants with wild-type 1 and 3 subunits resulted in functional GABARs. The GABA EC₅₀ values of the 13(M278S), 13(V279T), 13(S280I) and 132L(I282S) isoforms were similar to those of their respective wild-type receptors (Table 1), suggesting that these point mutations in the M2 triplet had little effect on GABA sensitivity.

The Zn²⁺ IC₅₀ values of GABARs containing any of the M2 triplet mutants were similar to those of GABARs containing wild-type subunits (Fig. 6A and B). The 13(M278S), 13(V279T) and 13(S280I) isoforms had average IC₅₀ values that were similar to that of 13 receptors (Table 2). The 132L(I282S) isoform had an average IC₅₀ that was similar to that of 132L receptors (Table 2). These results indicated that mutations of the individual residues in the M2 triplet did not replicate the effect of the triple mutation on the Zn²⁺ sensitivity of ternary GABARs.

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Figure 6. Zn2+ sensitivity of GABARs containing subunits with M2 triplet mutations A, representative whole-cell currents from L929 fibroblasts expressing 13(M278S) receptors (upper left), 13(V279T) receptors (upper right), 13(S280I) receptors (lower left), or 132L(I282S) receptors (lower right). GABA or GABA plus 10 M Zn2+ was applied for 7-12 s (horizontal bars) to cells voltage clamped at -75 mV. B, concentration-response curves for cells expressing 13(M278S) receptors (), 13(V279T) receptors (), 13(S280I) receptors (), or 132L(I282S) receptors () are shown by continuous lines. Concentration-response curves for wild-type 13 (left) and 132L (right) receptors are shown for comparison (dashed lines). The peak response to each concentration of Zn2+ was normalized as a percentage of the maximum current response to GABA alone for each cell. Values are means ± S.E.M. Data for each isoform were fitted with a four-parameter logistic equation with the indicated IC50 values and Hill slopes (nH).

The M2-M3 loop. The M2-M3 loop is involved in modulating the Zn²⁺ sensitivity of receptors containing the 1 or 6 subunit subtypes (Fisher & Macdonald, 1998). 632L receptors are more sensitive to Zn²⁺ than 132L receptors. Residue H273 of the 6 subunit confers increased Zn²⁺ sensitivity to 6 subunit-containing receptors whereas the homologous residue in the 1 subunit, N274, confers decreased Zn²⁺ sensitivity to 1 subunit-containing receptors. In 112 receptors, 1(N274) has been shown to be water accessible (Xu & Akabas, 1996), and sequence alignment indicates that the position occupied by 6(H273) differs between the and subunit families (Fig. 7A). We hypothesized that the positively charged K285 in the 2L subunit might electrostatically repulse Zn²⁺ from the outer mouth of the channel whereas the polar, uncharged S283 residue in the subunit might interact with Zn²⁺ (Karlin & Zhu, 1997) and thereby stabilize the cation in the channel. It has been previously reported that replacement of K285 with an alanine residue did not change the Zn²⁺ insensitivity of ternary receptors (Smart et al. 1994). To assess the role of this M2-M3 residue in determining Zn²⁺ sensitivity, the point mutations S283K and K285S were made in the and 2L subunits, respectively.

Coexpression of or 2L M2-M3 mutant subunits with wild-type 1 and 3 subunits resulted in functional GABARs. The GABA EC₅₀ values of the two mutant subunit-containing isoforms, 13(S283K) and 132L(K285S), were similar to those of their respective wild-type receptors (Table 1). These results indicated that these point mutations in the M2-M3 loop had little effect on GABA sensitivity.

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Figure 7. Zn2+ sensitivity of GABARs containing M2-M3 mutant subunits A, M2-M3 loop sequences for the rat 1, 6, 2L and subunits are shown. Residue numbers are based on the mature protein amino acid sequences. Residues in the 1 and 6 subunits previously shown to influence Zn2+ sensitivities of 132L and 632L receptors are underlined (Fisher & Macdonald, 1998). The homologous residues in the 2L and subunits that were targeted for mutagenesis are in bold and italicized. B, representative whole-cell currents from L929 fibroblasts expressing 13(S283K) receptors (left) and 132L(K285S) receptors (right). The indicated concentrations of GABA were applied for 6-8 s (horizontal bars) to cells voltage clamped at -75 mV. C, concentration-response curves for cells expressing 13(S283K) receptors () and 132L(K285S) receptors (). Concentration- response curves for wild-type 13 (left) and 132L (right) receptors are shown for comparison (dashed lines). The peak response to each concentration of Zn2+ was normalized as a percentage of the maximum current response to GABA alone for each cell. Values are means ± S.E.M. Data for each isoform were fitted with a four-parameter logistic equation with the indicated IC50 values and Hill slopes (nH).

Mutations in the M2-M3 loop of or 2L subunits had different effects on the Zn²⁺ sensitivities of ternary GABARs (Fig. 7B and C). The 13(S283K) isoform had an average IC₅₀ that was slightly higher than that of 13 receptors (Table 2), suggesting that substitution of a lysine residue at S283 in the wild-type background had minimal effect on Zn²⁺ sensitivity. However, the 132L(K285S) isoform had an average IC₅₀ that was lower than that of 132L receptors (Table 2). This mutant subunit resulted in a reduction in Zn²⁺ sensitivity that was 15 % of the log difference between Zn²⁺ IC₅₀ values of 132L and 13 receptors, suggesting that substitution of a serine residue at K285 in the wild-type 2L subunit caused an increase in Zn²⁺ sensitivity. Similar to the M2 triple mutants, these point mutations in the M2-M3 loop of and 2L subunits did not have equivalent effects on Zn²⁺ sensitivity, suggesting that the subunit context was an added factor (see Discussion).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

For the purposes of this discussion, we defined a functional domain as one or more amino acids that determined the effectiveness of a modulatory compound (e.g. Zn²⁺). The functional domain could represent the actual binding site for the compound, a transduction device between binding and drug effect, or a structural feature that could indirectly influence binding and/or transduction. In our study, the functional domain was defined by functional differences (e.g. IC₅₀) that were introduced by chimera construction and mutagenesis. Using this approach we identified novel structural determinants of low Zn²⁺ sensitivity for 2L subunit-containing ternary GABARs (Fig. 8). One functional domain in the 2L subunit for low Zn²⁺ sensitivity was localized to a subunit region forming the outer vestibule of the channel and was composed of residues in the M2 segment and the M2-M3 loop. The other functional domain in the 2L subunit for low Zn²⁺ sensitivity was localized to the N-terminal extracellular region. Together, these two functional domains appear to form the basis for differences in Zn²⁺ sensitivity between and receptors.

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Figure 8. Schematic representation of two domains of the 2L subunit involved in conferring low Zn2+ sensitivity The putative membrane topology of a 2L subunit is shown. The extracellular N- and C-termini are indicated (N, C) and the four putative transmembrane segments labelled (M1-M4). The subunit domains involved in conferring low Zn2+ sensitivity to ternary GABARs are indicated by dashed lines with identified key residues (boxed text) shown in their approximate locations. The first domain (I) is composed of the N-terminal extracellular region. The second domain (II) is composed of a triplet of amino acid residues (STI) at the extracellular end of the M2 segment and a single residue (K) at the proximal end of the M2-M3 loop.

Role of the outer vestibule in regulating Zn²⁺ sensitivity

One functional domain in the 2L subunit conferring low Zn²⁺ sensitivity was localized to the region of the outer vestibule of the channel. It was composed of the extracellular end of the M2 segment and the proximal end of the M2-M3 loop. The 2L subunit determinant in the M2 segment for reduced Zn²⁺ sensitivity consisted of three amino acid residues rather than a single one as was found for the subunits (Wooltorton et al. 1997; Horenstein & Akabas, 1998). Individual substitutions within the triplet based on 2L and sequence differences did not change the Zn²⁺ sensitivity of ternary receptors. As in the 1 and 6 subunit subtypes (Fisher & Macdonald, 1998), the 2L subunit M2-M3 loop was also found to make a contribution to Zn²⁺ sensitivity. Although these two 2L subunit regions were identified as separate determinants, it is likely that they form a single functional domain that was inadvertently divided by the design of our chimeric subunits.

It is likely that the M2 triplet of 2L and subunits is not directly involved in Zn²⁺ binding. In the subunit family, the homologous residue to the first position in the triplet has been shown to influence sensitivity to a variety of modulatory compounds. Replacement of the 1 subunit subtype with either the 2 or 3 subunit subtype confers greater receptor sensitivity to furosemide inhibition as well as to loreclezole, ethanol, enflurane, etomidate and -carboline potentiation (Wingrove et al. 1994; Stevenson et al. 1995; Belelli et al. 1997; Mihic et al. 1997; Thompson et al. 1999). The differences in drug sensitivities have been attributed to N265 (rat 3 subunit) and the homologous S265 (rat 1 subunit). These observations suggest that the first position in the M2 segment of the 2L and GABAR subunits has a role in transduction of modulator effects rather than in direct binding. Transduction of Zn²⁺ binding may be a role subserved by the second and third positions of the M2 triplet as well as by 2L(K285) in the M2-M3 loop. Residues 2(T281) and 2(I282) have recently been identified as two of the three transduction elements required to couple benzodiazepine binding to GABA current potentiation (Boileau & Czajkowski, 1999). The third element that was identified is 2(S291), which is six amino acids C-terminal to 2(K285) (Boileau & Czajkowski, 1999).

The third position of the M2 triplet has been shown to largely determine the Zn²⁺ sensitivity of homomers and binary receptors (Wooltorton et al. 1997; Horenstein & Akabas, 1998). Substitution of a serine (as in the subunit) or an isoleucine (as in the 2L subunit) for the histidine in this subunit position was found to decrease the Zn²⁺ sensitivity of 11 GABARs (Horenstein & Akabas, 1998). Substitution of a histidine at the homologous position of the 1 subunit (S271) was found to increase the Zn²⁺ sensitivity of 11 receptors (Horenstein & Akabas, 1998). Thus, this position appears to be available for Zn²⁺ binding in and subunits. In 2L subunits, however, Zn²⁺ interaction does not seem plausible as substitution of a histidine at 2(I282) was not found to increase the Zn²⁺ sensitivity of 112 receptors (Horenstein & Akabas, 1998).

Role of the N-terminus in regulating Zn²⁺ sensitivity

Another determinant of the low Zn²⁺ sensitivity of 2L subunits was localized to the N-terminus. This result was somewhat unexpected in the light of recent studies pointing to the extracellular portion of the M2 segment and the M2-M3 loop as important determinants of GABAR Zn²⁺ sensitivity (Wooltorton et al. 1997; Fisher & Macdonald, 1998; Horenstein & Akabas, 1998). However, it was shown that recombinant GABA_C receptor 1 subunits possessed a determinant of Zn²⁺ sensitivity in the homologous extracellular region (Wang et al. 1995). A single residue, H156, was found to be critical for Zn²⁺ sensitivity. Residue H156 is homologous to a highly conserved asparagine, which is found among all GABAR subunit families except . Interestingly, this residue is adjacent to H101 (rat sequence) in the 1 subunit, which is required for benzodiazepine sensitivity in ternary receptors (Smith & Olsen, 1995). The amino acid sequence in the vicinity of this residue is highlighted by two tryptophan residues (W69 and W94 in the rat GABAR 1 subunit) that are conserved in all members of the ligand-gated ion channel superfamily and in GABARs demarcate GABA and benzodiazepine binding regions (Smith & Olsen, 1995). In fact, mutation of the 1(H156) has also been found to influence the GABA sensitivity of 1 homomers (Kusama et al. 1994).

Functional domains determining the Zn²⁺ sensitivity of another member of the ligand-gated ion channel superfamily, the glycine receptors (GlyRs), have also been localized to the extracellular N-terminus. Mutagenesis based on chimeric subunit analysis has identified amino acid residues influencing potentiation and inhibition of human 1 homomers by Zn²⁺ (Lynch et al. 1998; Laube et al. 2000). Replacement of D80 resulted in a loss of potentiation and replacement of T112 resulted in a loss of inhibition. These two residues are located near the conserved tryptophan residues. Mutagenesis based on histidine targeting has also identified residues influencing potentiation and inhibition of GlyR currents by Zn²⁺ (Harvey et al. 1999). Mutation of H107 abolished Zn²⁺-mediated inhibition and mutation of H215 abolished Zn²⁺-mediated potentiation. Mutation of H109, however, abolished both inhibition and potentiation by Zn²⁺. Histidines 107 and 109 are proximal to the conserved tryptophan residues whereas H215 is not.

The complex nature of functional domains for Zn²⁺ in the N-terminal extracellular region of GlyRs suggests that such domains may also be complex in the homologous region of the GABAR 2L and subunits. If the tertiary subunit of ternary GABARs participates in Zn²⁺ binding, then one might expect that the Zn²⁺ functional domains could be identified by looking for the presence or absence of common Zn²⁺ co-ordinating residues (H, C, D and E; Karlin & Zhu, 1997). This approach will not work, however, if the functional domains are involved in a role other than Zn²⁺ binding. Thus, chimeric subunit analysis would prove more fruitful.

Subunit context of mutations and shifts in Zn²⁺ sensitivity

The shifts in Zn²⁺ sensitivity conferred by our site-directed mutant subunits were asymmetric. The triple mutations in the M2 segment of the 2L subunit induced a 48 % shift towards subunit-like sensitivity whereas in the subunit, the mutations induced only a 17 % shift towards 2L subunit-like sensitivity. The point mutation in the M2-M3 loop of the 2L subunit induced a 15 % shift towards subunit-like sensitivity whereas in the subunit, the mutation induced only a 4 % shift towards 2L subunit-like sensitivity. Although it is tempting to presume that asymmetry is an indicator of irrelevance of a given residue or residues, we would argue that, in fact, it points to the importance of context (i.e. tertiary structure) for functional domains. In the 'native' context of the 2L subunit, removal of the M2 triplet and the M2-M3 loop residue can be interpreted as a loss of elements necessary for low Zn²⁺ sensitivity. In the 'non-native' context of the subunit, introduction of the 2L subunit M2 triplet and M2-M3 loop residue cannot, however, be interpreted as acquisition of these same elements because the appropriate subunit context is not available. Therefore, these mutations would not be expected to have the symmetrical effect on Zn²⁺ sensitivity in an otherwise 'non-native' subunit context.

The importance of subunit context for functional domain properties also was demonstrated by the shifts of Zn²⁺ sensitivity induced by the -2L chimeric subunits. Removal of the 2L subunit N-terminus (-2L(M1e)) resulted in a 63 % shift in sensitivity from 2L subunit-like to subunit-like. The estimated contribution of the N-terminus to the low Zn²⁺ sensitivity of 2L subunits based on the site-directed mutant data would indicate a shift of ~40 % (see above). Sequential removal of the M2 triplet (-2L(M2e)) and the M2-M3 loop residue (-2L(M3e)) resulted in shifts of ~20 % each from 2L subunit-like to subunit-like. The sum of these shifts (~40 %) is somewhat lower than the sum of those induced by the site-directed mutations involving these subunit segments where the subunit context included the 2L N-terminus (~60 %). Thus, the potency of the 2L subunit outer vestibule in lowering Zn²⁺ sensitivity appears to be reduced in the context of the subunit N-terminus.

Although our study demonstrated that the N-terminus of the 2L and GABAR subunits contributed to Zn²⁺ sensitivity, it would not be surprising if the homologous regions of and subunits were also found to play a role. The alignment of various N-terminal residues implicated in agonist and benzodiazepine binding among the different subunit families suggests that residues involved in Zn²⁺ inhibition are also aligned (Sigel & Buhr, 1997). Indeed, differences in the mechanism of Zn²⁺ antagonism (non-competitive, competitive, mixed) among various receptor isoforms (Legendre & Westbrook, 1991; Gingrich & Burkat, 1998; Krishek et al. 1998) might be explained by the availability of Zn²⁺ functional domains, not only near the channel mouth but also at subunit interfaces whereby agonist binding could be influenced.

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Acknowledgements

We thank Sharon Baughman, Hyun Chung, Nadia Esmaeil, José S. Santos, Lisa M. Sharkey, Fang Sun and Jie Zhang for technical assistance and Matt T. Bianchi for critical reading of the manuscript. This work was supported by National Institutes of Health grants R01-NS33300 (R.L.M.) and 5T32-NS07222 (N.N.) and an Epilepsy Foundation of America fellowship (N.N.).

Corresponding author

R. L. Macdonald: Neuroscience Lab Building, 1103 E. Huron Street, Ann Arbor, MI 48104-1687, USA.

Email: rlmacd{at}umich.edu

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