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  • Photoincorporation of pTFDBzox AP occurred

    2021-09-14

    Photoincorporation of 21-pTFDBzox-AP occurred predominantly in the β3 subunit, very modestly in the α1 subunit and not at all in the γ2 subunit. These observations taken together with the sensitivity of 21-pTFDBzox-AP enhancement of GABA currents to the α1 M1 mutation Q242 W4 suggest that the 21-pTFDBzox-AP binding site is in the β3+ – α1– subunit interface. Support for steroid interaction on the β+ side of the interface comes from two studies. First, Chen et al., [17] using β3 homopentamers and the photolabel 5 (6-AziP) identified β3 F301 on the third transmembrane helix M3, on the plus side of the β– β interface, as a photoincorporation site, but were unable to establish its pharmacological specificity. Second, a recent substituted cysteine and modification of protection (SCAMP) protocol mapped alphaxalone–contacting residues to the cytoplasmic end of the β3-subunit's M3, including β3 F301 in α1β3γ2L receptors [30]. Unfortunately, α1 M1 Q242W, on the minus side of the β3+ – α1– subunit interface, is not amenable to SCAMP studies, so evidence for this interface in heteromeric receptors rests largely on the original mutagenesis study. However, two recent crystallography studies probed interactions of a number of natural steroid ligands with the transmembrane domain of α1 and α5 subunits in the unnatural homomeric chimeras consisting of GLIC ECD – human α1 TMD [15] and human β3 ECD – α5 TMD [16]. These studies show that steroids bind at the cytoplasmic end of the transmembrane domain in the α+ – α– subunit interface. Of particular interest, the 3α hydroxyls of THDOC (3) and pregnanolone (1) form a ZD 7288 sale with α1 Q242 and α5 Q245 on M1 on the minus side of the interface [15,16]. This glutamine is conserved in all six α-subunits but is a tryptophan in the β3 and γ2, and indeed in all other GABAAR subunits. This suggests that the α– interaction may be required for high affinity steroid interaction, but the β3 homopentamer photolabeling study implies that this requirement is not absolute. The effective concentrations could not be established in the structural studies because the THDOC enhancement curve for the GLIC ECD – human α1 TMD chimera does not saturate, and the β3 ECD – α5 TMD receptor was spontaneously open. Finally, in the GLIC ECD – human α1 TMD study [15], the inhibitory steroid pregnanolone sulfate interacted only with the transmembrane domain of the α1 subunit and its binding pocket did not overlap with that of THDOC, an observation that is consistent with our finding that pregnenolone sulfate does not bind to the 21-pTFDBzox-AP site. In our previous steroid photolabeling study [22], we found that photolabeling of α1β3γ2 receptor by ligands 12 and 13 was not inhibited by other steroid ligands. We noted that the photoactive moieties had limited mobility relative to the steroid backbone, leading us to hypothesize that the steroid backbone of those photolabels binds with its upper edge pointing out of the binding site, as previously suggested [32]. In this work, we found that substitutions on the lower side of the ring (compounds 14 and 15) caused a significant loss of potency, suggesting that the lower edge of the steroid backbone binds with its receptor site so that even modest substitutions in the 6-position disrupt binding. Consequently, compounds 6 and 14, which have a diazirine on the upper and lower side of the steroid ring system respectively, modulate agonist binding with potencies of 0.25 and 12 μM, respectively. The crystallography studies referred to above are quite consistent with this binding pose of steroids in their site.
    Conclusions We have synthesized and characterized the first steroid photolabel, 21-pTFDBzox-AP, to photoincorporate into heteromeric human GABAARs with pharmacological specificity. It neither binds in the etomidate site nor the R–mTFD-MPAB site, which are at the extracellular end of the transmembrane domain β+ – α– and γ+ – β– subunit interfaces, respectively. It photolabels almost exclusively into the β-subunit, and based on the evidence discussed above likely binds in the β+ – α– interface. Most imortantly, [3H]21-pTFDBzox-AP is well positioned to quantitatively probe binding of ligands to the steroid site in human GABAARs of any subunit composition (see for example Table 1) in a way that crystallography and mass spectrometry (a least to date) would find difficult.