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Keywords:

  • GABAA receptor;
  • channel;
  • modulation;
  • neurosteroid

Background and purpose:  Potentiating neurosteroids are some of the most efficacious modulators of the mammalian GABAA receptor. One of the crucial interactions may be between the C20 ketone group (D-ring substituent at C17) of the neurosteroid, and the N407 and Y410 residues in the M4 domain of the receptor. In this study, we examined the contribution of hydrogen bonding between 17β-substituents on the steroid D-ring and the GABAA receptor to potentiation by neurosteroids.

Experimental approach:  Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing wild-type and mutant α1β2γ2L GABAA receptors.

Key results:  A steroid with a 17β-carbonitrile group (3α5α18nor17βCN) was a potent and efficacious potentiator of the GABAA receptor. Potentiation was also shown by a cyclosteroid in which C21 and the C18 methyl group of (3α,5α)-3-hydroxypregnan-20-one are connected within a six-membered ring containing a double bond as a hydrogen bond acceptor (3α5αCDNC12), a steroid containing a 17β-ethyl group on the D-ring (3α5α17βEt) and a steroid lacking a 17β-substituent on the D-ring (3α5α17H). Single-channel kinetic analysis indicates that the kinetic mechanism of action is the same for the neurosteroid 3α5αP, 3α5α18nor17βCN, 3α5αCDNC12, 3α5α17βEt and 3α5α17H. Interestingly, 3α5α17βEt, at up to 3 µM, was incapable of potentiating the α1N407A/Y410F double mutant receptor.

Conclusions and implications:  Hydrogen bonding between the steroid 17β-substituent and the GABAA receptor is not a critical requirement for channel potentiation. The α1N407/Y410 residues are important for neurosteroid potentiation for reasons other than hydrogen bonding between steroid and receptor.