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 t he 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 no rmal levels of specific surface [H-3]granisetron ([H-3]BRL-43694) binding s ites. To determine what properties of the conserved proline are critical fo r the function of the channel, two imino acids and an alpha-hydroxy acid we re incorporated at the proline position using the nonsense suppression meth od. trans-3-Methyl-proline, pipecolic acid, and leucic acid were able to re place the conserved proline to produce active channels with EC50 values sim ilar to that for the wild-type receptor. These trends are preserved in the heteromeric receptors consisting of 5-HT3A and 5-HT3B subunits in oocytes. The prominent common feature among these residues and proline is the lack o f hydrogen bond donor activity, potentially resulting in a flexible seconda ry structure in the M1 region. Thus, lack of hydrogen bond donor activity m ay be a key element in channel gating and may explain the high degree of co nservation of this M1 proline.