Abstract
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 [3H]granisetron ([3H]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 α-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 EC50 values similar 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 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.
Footnotes
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Send reprint requests to: Dr. Henry A. Lester, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. E-mail: lester{at}caltech.edu
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This work was supported by National Institutes of Health Grants MH49176, NS34407, and NS10305.
- Abbreviations:
- M1
- transmembrane domain 1
- 5-HT
- 5-hydroxytryptamine (serotonin)
- 5-HT3A
- 5-HT3B, serotonin receptor type 3 A, B subunits
- m5-HT3A-UAG
- mouse serotonin receptor type 3 A mRNA containing the UAG stop codon rather than the M1 Pro256 codon
- nAChR
- nicotinic acetylcholine receptor
- tRNA-THG73-Xaa
- tRNA THG73 acylated with amino or hydroxy acid
- TMB-8
- 8-(diethylamine)octyl-3,4,5-trimethoxybenzoate
- P3m
- trans-3-methyl-proline
- Pip
- pipecolic acid
- Lah
- leucic acid
- Received December 9, 1999.
- Accepted February 12, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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