TY - JOUR
T1 - The molecular determinants of neurosteroid binding in the GABA(A) receptor
AU - Sugasawa, Yusuke
AU - Bracamontes, John R.
AU - Krishnan, Kathiresan
AU - Covey, Douglas F.
AU - Reichert, David E.
AU - Akk, Gustav
AU - Chen, Qiang
AU - Tang, Pei
AU - Evers, Alex S.
AU - Cheng, Wayland W.L.
N1 - Publisher Copyright:
© 2019 Elsevier Ltd
PY - 2019/9
Y1 - 2019/9
N2 - Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.
AB - Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.
KW - ELIC
KW - GABA receptor
KW - Mass spectrometry
KW - Mutation
KW - Neurosteroid
KW - Photoaffinity labeling
UR - http://www.scopus.com/inward/record.url?scp=85067076543&partnerID=8YFLogxK
U2 - 10.1016/j.jsbmb.2019.105383
DO - 10.1016/j.jsbmb.2019.105383
M3 - Article
C2 - 31150831
AN - SCOPUS:85067076543
SN - 0960-0760
VL - 192
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
M1 - 105383
ER -