Mutagenesis and computational docking studies support the existence of a histamine binding site at the extracellular beta 3+beta 3-interface of homooligomeric beta 3 GABA(A) receptors

作者：Hoerbelt Paul^*; Ramerstorfer Joachim; Ernst Margot; Sieghart Werner; Thomson Jeffrey L; Hough Lindsay B; Fleck Mark W

来源：Neuropharmacology, 2016, 108: 252-263.

DOI：10.1016/j.neuropharm.2016.04.042

• Histamine
• GABA(A) receptor
• Ligand-gated ion channel
• Bicuculline
• Mutagenesis
• Docking

Histamine is an important neurotransmitter that exerts its physiological actions through H1-4 metabotropic receptors in mammals. It also directly activates ionotropic GABA(A) receptor (GABA(A)R) beta 3 homooligomers and potentiates GABA responses in alpha beta heterooligomers in vitro, but the respective histamine binding sites in GABA(A)Rs are unknown. We hypothesized that histamine binds at the extracellular beta+beta- interface at a position homologous to the GABA binding site of heterooligomeric GABA(A)Rs. To test this, we individually mutated several residues at the putative ligand binding minus side of a rat GABA(A)R beta 3 wild type subunit and of a beta 3 subunit that was made insensitive to trace Zn2+ inhibition [beta 3(H267A); called (Z)beta 3]. (Z)beta 3, (Z)beta 3(Y62L), (Z)beta 3(Q64A), (Z)beta 3(Q64E), alpha 1(Z)beta 3, or alpha 1(Z)beta 3(Y62L) receptors were studied in HEK293T cells using whole cell voltage clamp recording. beta 3, beta 3(Y62C), beta 3(Q64C), beta 3(N41C), beta 3(D43C), beta 3(A45C) or beta 3(M115C) receptors were examined in Xenopus oocytes using two-electrode voltage clamp. Histamine directly activated (Z)beta 3 and beta 3 homooligomers and potentiated GABA actions in alpha 1(Z)beta 3 heterooligomers. Receptors containing Z beta 3(Y62L), beta 3(Y62C) and beta 3(D43C) showed markedly reduced histamine potency, but homo- and heterooligomers with z133(Q64E) exhibited increased potency. The GABA(A)R alpha beta(gamma) competitive antagonist bicuculline elicited sub-maximal agonist currents through (Z)beta 3 homooligomers, the potency of which was strongly decreased by (Z)beta 3(Y62L). Mutations beta 3(N41C), beta 3(A45C) and beta 3(M115C) disturbed receptor expression or assembly. Computational docking into the crystal structure of homooligomeric beta 3 receptors resulted in a histamine pose highly consistent with the experimental findings, suggesting that histamine activates beta 3 receptors via a site homologous to the GABA site in alpha beta gamma receptors.

• 出版日期2016-9