[Frontiers in Bioscience 1, d214-233, September 1, 1996]
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MOLECULAR BIOLOGY OF THE GABAA RECEPTOR: FUNCTIONAL DOMAINS IMPLICATED BY MUTATIONAL ANALYSIS

Martin Davies1, Alan N. Bateson1,2 and Susan M. J. Dunn1,2

1 Department of Pharmacology

2Division of Neuroscience, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Received 07/16/96; Accepted 07/22/96; On-line 09/01/96

5. MUTATIONAL ANALYSIS OF THE CHANNEL FORMING REGION

Hydropathy plots of each LGIC subunit reveal four conserved hydrophobic domains that are predicted to be transmembrane (14-17). Of these four regions, the second transmembrane domain, referred to as TM2, appears to form the lining of the ion channel. Extensive photolabelling studies and mutational analysis of the nAChR have identified this region as being involved in ion channel formation (for comprehensive reviews see 91, 92). Furthermore, synthetic peptides corresponding to the TM2 domain of the nAChR (93), and more recently of the GlyR alpha subunit (94), have been shown to form ion channels in lipid bilayers.

A recently developed technique, the substituted cysteine accessibility method (SCAM), has been used to identify amino acids that are exposed to the hydrophilic lumen of ion channels (95). This technique is based on replacing amino acids in the putative channel forming region with cysteines and then looking at the accessibility of the residues to modification by sulfhydryl-reactive agents. It is assumed that any residue that is exposed to the channel lumen will be accessible to the modifying agents, and modification will be detected as a change in the channel properties of the receptor. Furthermore, by examining the interval of the residues that are exposed to the modification agents, the secondary structure of the region may be predicted.

Application of SCAM to the GABAAR has identified likely channel-lining residues in the rat a1 subunit (96, 97, 98). Residues found to be accessible to cysteine-modifying agents correspond to some of those similarly identified by this technique in the nAChR a subunit (95, 99; see below and Figure 3). Residues V257, T261, T268 and I271, which lie towards the extracellular end of the TM2 domain were shown to be accessible to modifying agents in both the presence and absence of agonist, conditions which are presumed to reflect the closed and open state of the channel respectively. This observation suggests that the channel "gate" must lie deeper in the lumen than residue 257, which is deeper into the channel than had previously been suggested from the results of mutational studies of the nAChR. Coapplication of picrotoxin and GABA protected the mutated V257 from modification. The authors suggest that this may be a result of steric occlusion of the channel by picrotoxin, or less likely, from picrotoxin exerting an allosteric effect on the channel from a distant site.

CYTTM2EXT
rat GABAA 1
ESVPARTVFGVTTVLTMTTLSISARN
rat GABAA 2
DASAARVALGITTVLTMTTINTHLRE
rat GABAA 2
DAVPARTSLGITTVLTMTTLSTIARK
hum 1
RAVPARVPLGITTVLTMSTIITGVNA
hum 2
RAVPARVSLGITTVLTMTTIITGVNA
dros 1
NATPARVALGVTTVLTMTTLMSSTNA
gly 1
DAAPARVGLGITTVLTMTTQSSGSRA
gly 2
DASAARVPLGIFSVLSLASECTTLAA
5HT3
AETIFIVQLVHKQDLQRPVPDWLRHL
AchR 1
DS-GEKMTLSISVLLSLTVFLLVIVE
1   2  3    4  5   6     7

Figure 3. The TM2 region of several ligand gated ion channels. CYT, cytoplasmic face; EXT, extracellular face. Blue residues: 1, identified by SCAM as present in the channel lumen; 2, residue conferring high affinity loreclezole binding. Red residues: 1 and 2, residues conferring Picrotoxin sensitivity to rho receptors; drosophila 1, residue changed to a serine in insecticide-resistant strains.
Numerals correspond to the following : 1. Inner ring, 2. Intermediate ring, 3. Threonine ring, 4. Serine ring, 5. Leucine ring, 6. Valine ring, 7. Outer ring

The accessibility of modified residues to the cationic sulfhydryl reactive agent methanethiosulfonate ethyl-ammonium (MTSEA+) has suggested that the ion selectivity filter must lie deeper into the channel than residue 261. This contradicts earlier models in which the filter had been proposed to be formed by rings of negatively charged residues predicted to be present at the mouth of the channel (reviewed in 91; see Section 6). However, the SCAM data are consistent with the results of Galzi et al. (100) who found that substitution of the negatively charged residues in the putative intermediate and outer rings of the nAchR with neutral amino acids did not change the ion selectivity of the receptor. It is interesting that, in the latter study, one of the most important determinants of charge selectivity was shown to be the length of the linker between TMI and TM2, a stretch of amino acids that is predicted to be intracellular.

Investigation of the nAChR ion channel by SCAM has identified several residues in the TM2 region of the alpha subunit that are likely to be involved in its formation (see Figure 3). Initially, this method produced a pattern of cysteine modification in the nAChR subunit that was believed to be consistent with a ß-sheet structure for TM2 (95). However, further analysis suggested that this region exists as an alpha-helix interrupted by three amino acids in an extended ß-strand structure (99). A similar structure has now been proposed for the TM2 region of the GABAAR alpha1 subunit, with the interruption of the alpha-helix occurring in the region of T262 (98).

Examination of GABA receptors formed from rho1 and rho2 subunits points to one particular amino acid in the TM2 region of these subunits playing an important role in determining sensitivity to picrotoxin (101). When expressed in Xenopus oocytes, homomeric receptors formed by rho1 subunits are approximately ten-fold more sensitive to picrotoxin than their rho2 counterparts. The Hill coefficients of 1 for the rho1 subunit homomeric receptor and 2 for the rho2 subunit receptor, suggested that picrotoxin binding is non-cooperative in the former case and cooperative in the latter. Construction of chimaeras between these two subunits showed that the structural element conferring both the increased sensitivity of the rho2 subunit to picrotoxin and the change in Hill coefficient occurs at position 309 in the TM2 domain. At this position there is a proline in the rho1 subunit and a serine in the rho2 subunit. This residue occurs in the homologous position to V257 of the rat GABAAR alpha1 subunit identified using SCAM, and an alanine in the Drosophila alpha subunit which, when replaced by a serine in insects, confers resistance to picrotoxin and cyclodiene insecticides (102).

As for nAChR and other LGICs, mutational analysis of the TM2 region of the GABAAR provides strong evidence for this domain being part of the ion channel. Comparison of the primary sequences of various LGICs reveals conservation of residues both in TM2 and in the regions flanking this domain. Thus, the channel structure itself seems to be highly conserved even though some channels conduct cations, while others are anion selective. While a great deal of evidence points to the importance of the TM2 domain in channel formation, there is also evidence that regions other than TM2 are involved. Substitution of cysteine residues in the TM4 region of the nAChR, for example, can result in dramatic changes in the properties of ion conductance (103-105), suggesting that the overall conformation of the protein and its interaction with the lipid environment may also influence the properties of the ion channel.

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