Biol. Bull. 207: 156. (October 2004)
© 2004 Marine Biological Laboratory
The Second Transmembrane Domain 7' Position Influences Channel Kinetics in the Glycine 
1 Receptor
Eric B. Gonzales1,2 and
Glenn Dillon1
1 University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas
2 Grass Fellow 2004, Marine Biological Laboratory, Woods Hole, Massachusetts
The cys-loop family of ligand-gated ion channels (LGICs) consists of the nicotinic acetylcholine, serotonin type-3 (5-HT3), glycine, and GABAA receptors and is responsible for mediating rapid neurotransmission in the CNS and PNS. Several amino acid residues of the second transmembrane domain (TM2) have been shown to influence gating kinetics in members of the cys-loop superfamily. Of these receptors, the 5-HT3A and glycine receptor can form homomeric receptors. The homomeric 5-HT3A receptors have significantly slower gating kinetics. Our lab has shown that the TM2 L7'T mutation in the TM2 domain alters gating of the 5-HT3A receptor. We hypothesize that the TM2 7' residue influences channel gating in the homomeric glycine 
1 receptor. To test this hypothesis, we have systematically mutated the 7' residue to other amino acids to assess the properties required to alter gating. Wild type and mutant Gly 1 receptors were transiently expressed in HEK 293T cells and subsequently analyzed using the whole cell patch clamp recording technique. Concentration response profiles for these receptors were determined. Glycine sensitivity is altered in each of the mutant receptors tested, differing from the wild type 29.2 µM. The Gly EC50 for the T7'L and T7'A mutations, in µM, are 16.9 and 777, respectively. Using a rapid solution exchange system, the activation kinetics of these receptors are significantly slower at the glycine EC50 concentration. The T7'L mutation exhibits slower activation and deactivation than the wild-type receptor. These results demonstrate that the TM2 7' residue is a critical determinant of channel gating kinetics in glycine receptors and other cys-loop families of ion channels. Understanding how these receptors function will, in time, lead to the development of novel pharmacological therapeutics.
