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Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP.
Price KL
,
Lillestol RK
,
Ulens C
,
Lummis SC
.
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Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor.
Figure 1. Varenicline bound to
5-HTBP. (A) Location of varenicline (green)
at the interface between two subunits in the orthosteric 5-HTBP binding
site. (B) Alignment of 5-HTBP, AChBP, and the extracellular domain
of the 5-HT3A receptor subunit showing the approximate
location of the A–E binding loops. The residues mutated in
this study are highlighted in purple, and the residues that differ
between 5-HTBP and AChBP in yellow. (C) The 5-HTBP binding pocket
showing the orientation of varenicline (green) and nearby residues
on the principal (blue) and complementary (magenta) faces. The corresponding
5-HT3 receptor residues are in parentheses. (D) Hydrogen
bonds are present between varenicline and residues Y91, Y193, and
W145 on the principal face and residues I104 and I116 on the complementary
face via a water molecule.
Figure 2. HEK cell data. (A) Typical traces from HEK cells transfected with
WT 5-HT3 receptor cDNA, loaded with membrane potential
dye, and stimulated at 20 s with various concentrations of varenicline. F = arbitrary fluorescent units. (B) Concentration–response
curves constructed from FlexStation responses to 5-HT (squares, filled
line) and varenicline (circles, dashed line). Data = mean ± SEM, n = 4.
Figure 3. Concentration
response curves for WT and mutant 5-HT3 receptors expressed
in oocytes. Concentration response curves for
(A) 5-HT3A L184A and (B) 5-HT3A W90Y mutant
receptors (gray lines) compared to WT 5-HT3A receptors
(black lines) for 5-HT (solid lines) and varenicline (dashed lines)
reveal differences in EC50 and Rmax/Rmax,5-HT. Data = mean ±
SEM, n = 3–6.
Figure 4. The 5-HT3 receptor loop E region. Structural data from
the mouse 5-HT3A receptor structure (PDB: 4PIR) reveal a network
of hydrogen bonds involving the loop E aromatic residues Y141, Y143,
and Y153. Functional data show the importance of these residues for
proper functioning of the receptor.
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