Neuropharmacology 43 (2002) 669–678 www.elsevier.com/locate/neuropharm Tryptophan scanning mutagenesis in TM4 of the GABA A receptor a1 subunit: implications for modulation by inhaled anesthetics and ion channel structure Andrew Jenkins a,b,* , Alyson Andreasen a , James R. Trudell c , Neil L. Harrison a,b a Department of Anesthesiology, Weill Medical College of Cornell University, New York, NY 10021, USA b Department of Pharmacology, Weill Medical College of Cornell University, New York, NY 10021, USA c Department of Anesthesiology, Stanford University, Stanford, CA 94305, USA Received 10 May 2002; received in revised form 22 July 2002; accepted 23 July 2002 Abstract Previous studies have shown that amino acid residues in trans-membrane (TM) segments 1, 2 and 3 of the a subunit are critical for the enhancement of GABA A receptor function by inhaled anesthetics. In this study we used tryptophan (Trp) scanning mutagenesis between Ile 406 and Asn 417 in the a1 subunit to determine the effects of Trp substitution in the fourth transmembrane segment (TM4) on receptor gating and anesthetic modulation. Wild-type and mutant a1 subunits were transiently expressed in HEK 293 cells with wild-type b2 and g2s subunits and GABA-activated currents were recorded using whole-cell voltage clamp. The potentiation by three inhaled anesthetics (isoflurane, halothane and chloroform) of responses elicited by a submaximal concentration of GABA were also examined. EC 50 values for GABA at the mutant receptors were in the range 4–60 μM (wild-type=20 μM), indicating that Trp substitution can alter the apparent affinity of the receptor for GABA positively or negatively, dependent on position. The variation of the calculated EC 50 value for GABA exhibited an interesting periodicity, with the cycle length for each repeat corresponding to approxi- mately 3.6 amino acids. These data are consistent with an a-helical structure for the TM4 segment of the a subunit. Several of these Trp point mutations altered the ability of one or more of the three inhaled anesthetics to modulate receptor function; four of the 12 mutations abolished receptor modulation by one or more of the anesthetics tested. These data are consistent with a role for these residues at the extracellular end of TM4 in anesthetic modulation of GABA A receptors.  2002 Elsevier Science Ltd. All rights reserved. 1. Introduction GABA A receptors (GABA A -Rs) subserve fast synaptic inhibition mediated by g-aminobutyric acid (GABA), the most common inhibitory neurotransmitter in the CNS. GABA A -Rs are ligand-gated ion channels, constructed from five subunits; to date, a total of 20 subunits have been cloned (Whiting, 1999). While this extensive het- erogeneity potentially enables the assembly of over half a million different receptor subtypes, the variability * Corresponding author. C.V. Starr Laboratory for Molecular Neuropharmacology, Dept. Anesthesiology A1050, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, Box 124, New York, NY 10021, USA. Tel.: +1 212 746 1152; fax: +1 212 746 4879. E-mail address: anj2005@med.cornell.edu (A. Jenkins). 0028-3908/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII:S0028-3908(02)00175-2 exhibited in the mammalian brain is fortunately rather more restricted, with just over 20 subunit combinations accounting for ~99% of the GABA A -Rs in the CNS (McKernan and Whiting, 1996). The most prevalent receptor subtype in synapses of the adult mammalian CNS (accounting for ~40% of the total complement of GABA A -Rs) is a1b2g2 (McKernan and Whiting, 1996; Fritschy and Mo ¨hler, 1996). These receptors appear to assemble in a 2a:2b:1g stoichiometry (Chang et al., 1996; Farrar et al., 1999). Neuronal GABA A -R function is modulated by most general anesthetics (Krasowski and Harrison, 1999) at concentrations consistent with their pharmacological use (Franks and Lieb, 1994). For example, halothane (350 μM) prolongs inhibitory postsynaptic currents in pyrami- dal cells and interneurons in slices of rat hippocampus (Nishikawa and MacIver, 2000; Pearce et al., 1989).