Korean J Anesthesiol 2011 February 60(2): 109-118 Experimental Research Article DOI: 10.4097/kjae.2011.60.2.109 The effects of midazolam and sevoflurane on the GABAA receptors with alternatively spliced variants of the γ2 subunit Woosik Eom1, Jung Min Lee2, Jeongmi Park2, Kyungho Choi3, Sung­Jun Jung4, and Hee­Soo Kim2 1Department of Anesthesiology and Pain Medicine, National Cancer Center, Goyang, 2Department of Anesthesiology and Pain Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, 3Specific Organs Cancer Branch, Research Institute, National Cancer Center, Goyang, 4Department of Physiology, Hanyang University College of Medicine, Seoul, Korea Background: Emergence agitation after sevoflurane anesthesia in children can be prevented by midazolam. Alternative splicing of the GABAA receptor changes with age. Therefore, we hypothesized that alternative splicing of the γ2 subunit affects the GABA current when applying sevoflurane and midazolam. Methods: We performed the whole­cell patch clamp technique on human embryonic kidney 293 cells that were transfected with α1β2γ2L or α1β2γ2S. The concentration­response relations were recorded for midazolam and sevoflurane, and the co­application responses were measured at concentrations of 1.5 nM, 15 nM and 300 nM of midazolam and 0.5%, 2.0% and 4.0% of sevoflurane. Each GABA current was compared with that produced by 5 μM of GABA. Results: The concentration­response relationships for midazolam and sevoflurane were dose­dependent without any differences between the α1β2γ2L and α1β2γ2S subtypes. 1.5 nM and 15 nM of midazolam did not significantly enhance the current after treatment with 0.5% sevoflurane for both subtypes. The current after treatment with 2.0% sevoflurane was enhanced by 1.5 nM midazolam for the α1β2γ2S subtype, but not for the α1β2γ2L subtype. In the case of 2.0% sevoflurane with 15 nM of midazolam, and 4.0% sevoflurane with 300 nM of midazolam, the GABA currents were significantly enhanced for both subtypes. Conclusions: These results show that the difference in the γ2 subunit cannot explain the emergence agitation after sevoflurane anesthesia in children in vitro. This suggests that co­application of sevoflurane and midazolam enhances the GABA current according to the alternative splicing of the γ2 subunit and the concentration of both drugs. (Korean J Anesthesiol 2011; 60: 109­118) Key Words: Agitation, Alternative splicing, GABAA receptor, Gamma 2 subunit, Midazolam, Sevoflurane. Received: August 5, 2010. Revised: August 19, 2010. Accepted: August 20, 2010. Corresponding author: Woosik Eom, M.D., Department of Anesthesiology and Pain Medicine, National Cancer Center, 323, Ilsan-ro, Goyang 410-769, Korea. Tel: 82-31-920-1703, Fax: 82-31-920-1463, E-mail: [email protected] CC This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright ⓒ the Korean Society of Anesthesiologists, 2011 www.ekja.org GABA receptor, midazolam and sevoflurane Vol. 60, No. 2, February 2011 Introduction receptor [10]. Sevoflurane is a volatile anesthetic that was developed The GABAA receptor mediates rapid inhibitory action by relatively recently, and it is now widely used and preferred for using GABA, which is a major inhibitory neurotransmitter of clinical anesthesia, and especially for pediatric anesthesia. the central nervous system. Activation of the GABAA receptor Sevoflurane has been reported to potentiate GABA­induced induces the opening of chloride channels and this increases currents at the GABAA receptor. Sevoflurane enables the rapid the inward chloride current. The hyperpolarization of the induction and quick recovery after inhalational anesthesia membrane of neurons then decreases the post­synaptic action when using it because sevoflurane possesses several favorable potential of the neuron [1]. properties, including low blood and tissue solubility, non­ As the GABAA receptor has specific binding sites for GABA, pungency and limited cardiorespiratory depression, which may barbiturate, benzodiazepines and the anesthetic steroids, which make sevoflurane desirable for use in infants and children [11]. induces sedation, hypnosis, amnesia or anesthesia through The quick recovery from sevoflurane is likely to be accompanied their binding, the GABAA receptor is considered to be a major by emergence agitation, which often occurs after administering target protein of anesthesia [2]. sevoflurane in children even when sufficient analgesia is The GABAA receptor is a complex of 5 subunits of 19 different provided [12,13]. subunits (α, β, γ, δ, ε, π, θ, ρ, etc.) in mammals. Multiple One of the trials to reduce the unpleasant effects during combinations from the different subunits make several subtypes emergence is to use midazolam as premedication. It has been of GABAA receptor. Most GABAA receptors consist of two α reported that premedication with oral midazolam is effective subunits, two β subunits and a γ subunit. The complexity of for decreasing the occurrence of emergence agitation without GABAA receptors is still under investigation and there exits the delaying the discharge from the post­anesthesia care unit and possibility of new combinations of subunits. Each combination so this is safe and suitable for outpatient surgery [14]. However, of different subunits has unique physiologic characteristics and the mechanism is still not clear, and it is not known whether the drug responses are different from each other [3]. sevoflurane and midazolam have the interaction at the level of The γ2 subunits of the GABAA receptor exist as a long type the GABAA receptor. (γ2L) and a short type (γ2S) by alternative splicing of RNA. The The mechanism of agitation was described in the model of γ2L subunits have another 8 amino acids (LLRMFSFK) in the Sachdev and Kruk as the result of decreased inhibitory signals intracellular loop and the phosphorylation by protein kinase C to the globus pallidus interna/substantia nigra pars or the (PKC) occurs in this region (Ser343) [4]. disinhibition of the thalamocortical neurons and brain stem It seems that the agents binding to the γ2 subunit may have neurons by the disturbance of neuronal circuits [15]. So, it is different effects according to the subtype. The total expression expected that the change of GABAA receptor activity may cause of the γ2 subunit is similar for all ages, but γ2L/γ2S increases or reduce emergence agitation. by the up­regulation of the γ2L subunit and γ2L becomes We postulated that alternative splicing of γ2 subunit is related dominant in mature synapses according to age [5,6]. with the occurrence of emergence agitation on the basis of the The distribution and pattern of GABAA receptors are various characteristics of midazolam, sevoflurane and the γ2 subunit; according to the region of the central nervous system and the sevoflurane binds to GABAA receptor, a benzodiazepine like type of neuron. This is also true for γ2L and γ2S [7,8]. The most midazolam prevents emergence agitation and binds to the α common pattern of GABAA receptor is the α1β2γ2 type, which is and γ subunits of the GABAA receptor, and alternative splicing 43% of all the GABAA receptors [9]. This implies that the diversity of the γ2 subunit is different according to age. Therefore, of the subtype variants and the distribution of GABAA receptors we performed the whole­cell patch clamp to the α1β2γ2L may affect the effect of anesthetics and induce different effects and α1β2γ2S GABAA receptors that are expressed on human from subject to subject. embryonic kidney (HEK) 293 cells with using midazolam and/ Midazolam is a popular sedative and sevoflurane is a most or sevoflurane. commonly used inhalational anesthetic. It is known that these drugs show their sedative, hypnotic or anesthetic actions Materials and Methods by binding to GABAA receptors. The GABAA receptor has the loci for binding to benzodiazepine, barbiturate and steroids. Transfection of the γ2 cDNA of the GABAA receptor to Midazolam activates the GABAA receptor by augmenting the HEK cells the binding of GABA to the receptor and midazolam directly activates the GABAA receptor at a high concentration. The γ2 HEK 293 cells are suitable for the transient expression of subunit is essential for binding benzodiazepine to the GABAA GABAA receptors and electrophysiological study [16]. Culturing 110 www.ekja.org Korean J Anesthesiol Eom, et al. of the HEK 293 cells and expressing GABAA cDNA in the HEK 1.5 mm, Harvard Apparatus Ltd., Edenbridge, Kent, UK). The 293 cells were performed as previously described [17]. resistance of the patch pipette was 4-6 M when it was filled The HEK 293 cells (CRL­1573; American Type Culture with intracellular solution (145 mM N­methyl­D­glucamine Collection) were cultured on glass cover slips in a solution HCl, 5 mM HEPES/KOH and 0.1 mM CaCl2, pH 7.2). containing minimum essential medium (MEM) supplemented The expression of GABA cDNA was confirmed by the develop­ with 10% heated fetal bovine serum, glutamine (4 mM), ment of intrinsic current, which was measured from the penicillin G (100 U/ml) and streptomycin sulfate (100 μg/ml). fluorescing HEK 293 cells, but not from the non­fluorescing Human γ2S cDNA was made from human γ2L cDNA (JC cells by the application of GABA (10 μM) and inhibition of the Biotech, Seoul, Korea) by deletion mutagenesis and its intrinsic current by the application of bicuculline (10 μM), sequence was confirmed by polymerase chain reaction. which is an antagonist of the GABAA receptor. The γ­aminobutyric acid type A receptor complementary In addition to the continuous bath perfusion with extracellular DNAs (the human γ2L or γ2S cDNA, the rat α1 cDNAs and the solution, solutions including GABA, sevoflurane or midazolam rat β2 cDNAs: the rat α1 and β2 cDNAs were generous gifts from were applied to the cells for 3 s at a rate of 10 ml/min using a Dr.