Probing the role of a conserved M1 proline residue in 5-hydroxytryptamine(3) receptor gating.
A conserved proline residue is found in the first transmembrane domain (M1) of every subunit in the ligand-gated ion channel superfamily. The position of this proline between the N-terminal extracellular agonist binding and the second transmembrane (M2) channel lining domains in the primary sequence suggests its possible involvement in the gating of the receptor. Replacing this proline with alanine, glycine, or leucine in the 5-hydroxytryptamine (5-HT)(3A) homomeric receptors expressed in Xenopus laevis oocytes resulted in the absence of 5-HT-induced whole-cell currents, although there were normal levels of specific surface [(3)H]granisetron ([(3)H]BRL-43694) binding sites. To determine what properties of the conserved proline are critical for the function of the channel, two imino acids and an alpha-hydroxy acid were incorporated at the proline position using the nonsense suppression method. trans-3-Methyl-proline, pipecolic acid, and leucic acid were able to replace the conserved proline to produce active channels with EC(50) values similar to that for the wild-type receptor. These trends are preserved in the heteromeric receptors consisting of 5-HT(3A) and 5-HT(3B) subunits in oocytes. The prominent common feature among these residues and proline is the lack of hydrogen bond donor activity, potentially resulting in a flexible secondary structure in the M1 region. Thus, lack of hydrogen bond donor activity may be a key element in channel gating and may explain the high degree of conservation of this M1 proline.
Dang
H
H
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
England
P M
PM
Farivar
S S
SS
Dougherty
D A
DA
Lester
H A
HA
D000818:Animals; D004594:Electrophysiology; D006860:Hydrogen Bonding; D015640:Ion Channel Gating; D051379:Mice; D016296:Mutagenesis; D009865:Oocytes / Q000502:physiology; D011392:Proline / Q000235:genetics / Q000502:physiology*; D011985:Receptors, Serotonin / Q000235:genetics / Q000502:physiology*; D044406:Receptors, Serotonin, 5-HT3; D014982:Xenopus laevis
D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011392:Proline