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The α5 GABAA receptor subunit confers resistance to isoflurane inhibition
International Congress Series 1283 (2005) 189 – 192
www.ics-elsevier.com
The a5 GABAA receptor subunit confers resistance to isoflurane inhibition B.A. Orser a,b,c,e,*, V.B. Caraiscos a, E.K. You-Ten b, V.Y. Cheng a, J.G. Newell c, J.F. MacDonald c,d a
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 b Department of Anesthesia, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 c Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 d Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 e Department of Anesthesia, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada, M4N 3M5
Abstract. The volatile anesthetic, isoflurane, potentiates and inhibits GABAA receptors (GABAARs) function at low and high concentrations, respectively. We examined whether the a subunit isoform influences isoflurane inhibition by studying currents generated by human recombinant a5/1h3g2L GABAARs. The subunit isoforms selected for the study mediate a tonic (a5) and synaptic (a1) inhibitory conductance in hippocampal pyramidal neurons. The a5 subunit conferred partial resistance to isoflurane blockade compared with the a1 subunit. The steady-state current was preferentially reduced compared with the peak current suggesting that isoflurane inhibited GABAAR function by stabilizing an agonist-bound, closed conformational state or by a use-dependent steric blocking mechanism. D 2005 Elsevier B.V. All rights reserved. Keywords: GABA; Isoflurane; Tonic inhibition; a5 subunit; Hippocampus
1. Introduction Most inhibitory neurotransmission is mediated by GABAA receptors (GABAARs). These hetero-pentamers are comprised of different subunits that exhibit distinct patterns of distribution and pharmacological properties. Low concentrations of the volatile anesthetic, * Corresponding author. Department of Physiology, Room 3318, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada, M5S 1A8. Tel.: +1 416 978 0574; fax: +1 416 978 4940. E-mail address: [email protected] (B.A. Orser). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.085
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B.A. Orser et al. / International Congress Series 1283 (2005) 189–192
isoflurane interact with specific amino acid residues and increase GABAARs function [1]. The subunit composition of GABAAR critically influences the potentiating effects of isoflurane [2]. High concentrations of isoflurane (~ 200 AM) directly gate channel opening while even higher concentrations (N250 AM) reduce GABAAR function [3,4]. We test the hypothesis that the a subunit isoform determines the sensitivity of GABAARs to isoflurane inhibition. In the hippocampus, two forms of GABAAR-mediated inhibition have been identified. These include a fast, transient synaptic conductance generated by postsynaptic GABAARs and a tonic conductance generated by low ambient concentrations of GABA in the extracellular space [5,6]. The tonic and synaptic conductances in hippocampal pyramidal neurons are activated by GABAARs with different subunit compositions [7,8]. The tonic conductance is generated by a5 subunit-containing GABAARs that are primarily segregated to extrasynaptic regions [9–13]. In contrast, postsynaptic GABAARs that generate conventional synaptic inhibition are thought to contain a1 or a2 but not a5 subunits. Here, we compare the extent of isoflurane inhibition of human recombinant a5h3g2L with a1h3g2L GABAARs. 2. Materials and methods The methods have been previously described in detail [14]. HEK 293 cells were grown on poly-d-lysine-coated dishes 3–6 h before transfection. Lipofectamine was used to transfect cells with human cDNAs for a5 or a1 subunits plus h3 and g2L in a 1:1:1 ratio. Conventional whole-cell currents were recorded under voltage clamp conditions ( 60 mV). The extracellular solution contained 140 mM NaCl, 1.3 mM CaCl2, 5.4 mM KCl, 25 mM HEPES, and 28 mM glucose, with pH adjusted to 7.4 using 1 M NaOH. Recording electrodes were filled with a solution that contained: 140 mM CsCl, 10 mM HEPES, 11 mM EGTA, 2 mM MgCl2, 1 mM CaCl2, 4 mM MgATP, and 2 mM TEA, with the pH adjusted to 7.3 using CsOH. A saturating concentration of GABA (600 AM) was applied for 20 s to allow currents to desensitize to a stable level. Desensitization is the process by which GABAARs enter a non-conductive conformation while remaining in an agonistbound state. Isoflurane (25 and 2500 AM) was pre-applied for at least 30 s prior to coapplication of GABA and isoflurane 2 s. Concentrations of isoflurane are also expressed as fraction of the minimum alveolar concentration (MAC) where MAC is defined as the concentration that is required to produce immobility in response to a noxious stimulus in 50% of subjects [15]. One MAC equivalent of isoflurane for mice was estimated to be 250 AM and MAC values are similar for mice and humans [16,17]. Results are presented as mean F S.E.M. 3. Results Currents evoked by GABA (600 AM) peaked then rapidly decayed to a steady-state (Fig. 1A). Isoflurane (250 AM) caused a similar reduction in the peak current (Ip) generated by a1h32L and a5h3g2L GABAARs (85.9 F 5.7% of control, n = 7 versus 84.5 F 4.1% of control, n = 6, respectively). The steady-state current (Iss) generated by a1h3g2L GABAARs was considerably more sensitive to inhibition by isoflurane (250 AM) compared with a5h3g2L GABAARs. The Iss/Ip ratio was decreased more for a1h3g2L (71.0 F 6.1% of control, n = 7) compared with a5h3g2L GABAARs (90.0 F 4.3% of control, n = 6, p b 0.05, Fig. 1B). Isoflurane (2500 AM) almost abolished
B.A. Orser et al. / International Congress Series 1283 (2005) 189–192
191
Fig. 1. (A) GABA-evoked currents recorded in the absence and presence of isoflurane (ISO, 250 and 2500 AM) have been previously shown (14, Copyright 2004 by the Society for Neuroscience). (B) The effects of isoflurane on Iss and Iss/Ip are summarized.
currents recorded from a1h3g2L GABAARs (1.0 F 2.1% of control, n = 5) whereas currents from a5h3g2L GABAARs were only partially inhibited (38.5 F 11.5% of control, n = 5, p b 0.05).
4. Discussion The a5 subunit conferred resistance to isoflurane inhibition when compared with the a1 subunit and isoflurane preferentially inhibited the steady-state current recorded from both groups of receptors. The latter finding suggests that isoflurane inhibits GABAARs by a use-dependent open channel blocking mechanism or by allosterically increasing GABAAR desensitization and stabilization of an agonist-bound, closed conformational state [18]. We favor an allosteric mechanism as it is consistent with reports that show sevoflurane and halothane increase the rate and extent of GABAAR desensitization [16,17]. Also, the a5 subunit confers resistance to desensitization [13] and possibly resistance to drugs that increase desensitization. An open channel blocker would preferentially inhibit receptors with a high open probability such as those containing the a5 subunit (which is inconsistent with our data). While the inhibitory effects were examined at clinically relevant but high concentrations of isoflurane (1 and 10 MAC), it has been proposed that isoflurane inhibition occurs at lower concentrations [19]. The dual effects of potentiation and inhibition occur simultaneously resulting in a net response. The relative resistance to inhibition conferred by the a5 subunit may contribute to the dramatic potentiation by isoflurane of a tonic conductance in hippocampal pyramidal neurons. In contrast, postsynaptic currents generated by a5 subunit-deficient GABAARs were abolished by high concentrations of isoflurane (2500 AM).
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Acknowledgements The study was supported by grants from the Canadian Institutes of Health Research, Canada Research Chair and the Canadian Anesthesiologists’ Society. References [1] A. Jenkins, et al., Evidence for a common binding cavity for three general anesthetics within the GABAA receptor, J. Neurosci. 21 (6) (2001) U7 – U10. [2] I. Belelli, et al., General anaesthetic action at transmitter-gated inhibitory amino acid receptors, Trends Pharmacol. Sci. 20 (12) (1999) 496 – 502. [3] M.I. Banks, R.A. Pearce, Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects, Anesthesiology 90 (1) (1999) 120 – 134. [4] S. Neumahr, et al., Dual action of isoflurane on the gamma-aminobutyric acid (GABA)-mediated currents through recombinant a(1)h(2)g(2L)-GABA(A)-receptor channels, Anesth. Analg. 90 (5) (2000) 1184 – 1190. [5] Q.Y. Liu, et al., Persistent activation of GABAA receptor/Cl( ) channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons, J. Neurophysiol. 84 (3) (2000) 1392 – 1403. [6] D. Bai, et al., Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons, Mol. Pharmacol. 59 (4) (2001) 814 – 824. [7] J.Y. Yeung, et al., Tonically activated GABAA receptors in hippocampal neurons are high-affinity, lowconductance sensors for extracellular GABA, Mol. Pharmacol. 63 (1) (2003) 2 – 8. [8] B.M. Stell, I. Mody, Receptors with different affinities mediate phasic and tonic GABAA conductances in hippocampal neurons, J. Neurosci. (2002). [9] J.M. Fritschy, H. Mohler, GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits, J. Comp. Neurol. 359 (1) (1995) 154 – 194. [10] I. Brunig, et al., Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro, J. Comp. Neurol. 443 (1) (2002) 43 – 55. [11] I. Mody, Distinguishing between GABA(A) receptors responsible for tonic and phasic conductances, Neurochem. Res. 26 (8–9) (2001) 907 – 913. [12] A.L. Scotti, H. Reuter, Synaptic and extrasynaptic g-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development, Proc. Natl. Acad. Sci. U. S. A. 98 (6) (2001) 3489 – 3494. [13] V.B. Caraiscos, et al., Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by a5 subunit-containing g-aminobutyric acid type A receptors, Proc. Natl. Acad. Sci. U. S. A. 101 (10) (March 9) (2004) 3662 – 3667. [14] V.B. Caraiscos, et al., Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane, J. Neurosci. 24 (39) (2004) 8454 – 8458. [15] E.I. Eger, L.J. Saidman, B. Brandstater, Minimum alveolar anesthetic concentration: a standard of anesthetic potency, Anesthesiology 26 (6) (1965) 756 – 763. [16] M. Nakahiro, et al., General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons, FASEB J. 3 (7) (1989) 1850 – 1854. [17] J. Wu, N. Harata, N. Akaike, Potentiation by sevoflurane of the gamma-aminobutyric acid-induced chloride current in acutely dissociated CA1 pyramidal neurones from rat hippocampus, Br. J. Pharmacol. 119 (5) (1996) 1013 – 1021. [18] B. Hille, Classical Mechanisms of Block in Ionic Channels of Excitable Membranes, 3rd ed., Sinauer Associates, Inc., Sunderland, MA, 2001. [19] G. Hapfelmeier, H. Schneck, E. Kochs, Sevoflurane potentiates and blocks GABA-induced currents through recombinant alpha1beta2gamma2 GABAA receptors: implications for an enhanced GABAergic transmission, Eur. J. Anaesthesiol. 18 (6) (2001) 377 – 383.
www.ics-elsevier.com
The a5 GABAA receptor subunit confers resistance to isoflurane inhibition B.A. Orser a,b,c,e,*, V.B. Caraiscos a, E.K. You-Ten b, V.Y. Cheng a, J.G. Newell c, J.F. MacDonald c,d a
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 b Department of Anesthesia, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 c Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 d Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 e Department of Anesthesia, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada, M4N 3M5
Abstract. The volatile anesthetic, isoflurane, potentiates and inhibits GABAA receptors (GABAARs) function at low and high concentrations, respectively. We examined whether the a subunit isoform influences isoflurane inhibition by studying currents generated by human recombinant a5/1h3g2L GABAARs. The subunit isoforms selected for the study mediate a tonic (a5) and synaptic (a1) inhibitory conductance in hippocampal pyramidal neurons. The a5 subunit conferred partial resistance to isoflurane blockade compared with the a1 subunit. The steady-state current was preferentially reduced compared with the peak current suggesting that isoflurane inhibited GABAAR function by stabilizing an agonist-bound, closed conformational state or by a use-dependent steric blocking mechanism. D 2005 Elsevier B.V. All rights reserved. Keywords: GABA; Isoflurane; Tonic inhibition; a5 subunit; Hippocampus
1. Introduction Most inhibitory neurotransmission is mediated by GABAA receptors (GABAARs). These hetero-pentamers are comprised of different subunits that exhibit distinct patterns of distribution and pharmacological properties. Low concentrations of the volatile anesthetic, * Corresponding author. Department of Physiology, Room 3318, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada, M5S 1A8. Tel.: +1 416 978 0574; fax: +1 416 978 4940. E-mail address: [email protected] (B.A. Orser). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.085
190
B.A. Orser et al. / International Congress Series 1283 (2005) 189–192
isoflurane interact with specific amino acid residues and increase GABAARs function [1]. The subunit composition of GABAAR critically influences the potentiating effects of isoflurane [2]. High concentrations of isoflurane (~ 200 AM) directly gate channel opening while even higher concentrations (N250 AM) reduce GABAAR function [3,4]. We test the hypothesis that the a subunit isoform determines the sensitivity of GABAARs to isoflurane inhibition. In the hippocampus, two forms of GABAAR-mediated inhibition have been identified. These include a fast, transient synaptic conductance generated by postsynaptic GABAARs and a tonic conductance generated by low ambient concentrations of GABA in the extracellular space [5,6]. The tonic and synaptic conductances in hippocampal pyramidal neurons are activated by GABAARs with different subunit compositions [7,8]. The tonic conductance is generated by a5 subunit-containing GABAARs that are primarily segregated to extrasynaptic regions [9–13]. In contrast, postsynaptic GABAARs that generate conventional synaptic inhibition are thought to contain a1 or a2 but not a5 subunits. Here, we compare the extent of isoflurane inhibition of human recombinant a5h3g2L with a1h3g2L GABAARs. 2. Materials and methods The methods have been previously described in detail [14]. HEK 293 cells were grown on poly-d-lysine-coated dishes 3–6 h before transfection. Lipofectamine was used to transfect cells with human cDNAs for a5 or a1 subunits plus h3 and g2L in a 1:1:1 ratio. Conventional whole-cell currents were recorded under voltage clamp conditions ( 60 mV). The extracellular solution contained 140 mM NaCl, 1.3 mM CaCl2, 5.4 mM KCl, 25 mM HEPES, and 28 mM glucose, with pH adjusted to 7.4 using 1 M NaOH. Recording electrodes were filled with a solution that contained: 140 mM CsCl, 10 mM HEPES, 11 mM EGTA, 2 mM MgCl2, 1 mM CaCl2, 4 mM MgATP, and 2 mM TEA, with the pH adjusted to 7.3 using CsOH. A saturating concentration of GABA (600 AM) was applied for 20 s to allow currents to desensitize to a stable level. Desensitization is the process by which GABAARs enter a non-conductive conformation while remaining in an agonistbound state. Isoflurane (25 and 2500 AM) was pre-applied for at least 30 s prior to coapplication of GABA and isoflurane 2 s. Concentrations of isoflurane are also expressed as fraction of the minimum alveolar concentration (MAC) where MAC is defined as the concentration that is required to produce immobility in response to a noxious stimulus in 50% of subjects [15]. One MAC equivalent of isoflurane for mice was estimated to be 250 AM and MAC values are similar for mice and humans [16,17]. Results are presented as mean F S.E.M. 3. Results Currents evoked by GABA (600 AM) peaked then rapidly decayed to a steady-state (Fig. 1A). Isoflurane (250 AM) caused a similar reduction in the peak current (Ip) generated by a1h32L and a5h3g2L GABAARs (85.9 F 5.7% of control, n = 7 versus 84.5 F 4.1% of control, n = 6, respectively). The steady-state current (Iss) generated by a1h3g2L GABAARs was considerably more sensitive to inhibition by isoflurane (250 AM) compared with a5h3g2L GABAARs. The Iss/Ip ratio was decreased more for a1h3g2L (71.0 F 6.1% of control, n = 7) compared with a5h3g2L GABAARs (90.0 F 4.3% of control, n = 6, p b 0.05, Fig. 1B). Isoflurane (2500 AM) almost abolished
B.A. Orser et al. / International Congress Series 1283 (2005) 189–192
191
Fig. 1. (A) GABA-evoked currents recorded in the absence and presence of isoflurane (ISO, 250 and 2500 AM) have been previously shown (14, Copyright 2004 by the Society for Neuroscience). (B) The effects of isoflurane on Iss and Iss/Ip are summarized.
currents recorded from a1h3g2L GABAARs (1.0 F 2.1% of control, n = 5) whereas currents from a5h3g2L GABAARs were only partially inhibited (38.5 F 11.5% of control, n = 5, p b 0.05).
4. Discussion The a5 subunit conferred resistance to isoflurane inhibition when compared with the a1 subunit and isoflurane preferentially inhibited the steady-state current recorded from both groups of receptors. The latter finding suggests that isoflurane inhibits GABAARs by a use-dependent open channel blocking mechanism or by allosterically increasing GABAAR desensitization and stabilization of an agonist-bound, closed conformational state [18]. We favor an allosteric mechanism as it is consistent with reports that show sevoflurane and halothane increase the rate and extent of GABAAR desensitization [16,17]. Also, the a5 subunit confers resistance to desensitization [13] and possibly resistance to drugs that increase desensitization. An open channel blocker would preferentially inhibit receptors with a high open probability such as those containing the a5 subunit (which is inconsistent with our data). While the inhibitory effects were examined at clinically relevant but high concentrations of isoflurane (1 and 10 MAC), it has been proposed that isoflurane inhibition occurs at lower concentrations [19]. The dual effects of potentiation and inhibition occur simultaneously resulting in a net response. The relative resistance to inhibition conferred by the a5 subunit may contribute to the dramatic potentiation by isoflurane of a tonic conductance in hippocampal pyramidal neurons. In contrast, postsynaptic currents generated by a5 subunit-deficient GABAARs were abolished by high concentrations of isoflurane (2500 AM).
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B.A. Orser et al. / International Congress Series 1283 (2005) 189–192
Acknowledgements The study was supported by grants from the Canadian Institutes of Health Research, Canada Research Chair and the Canadian Anesthesiologists’ Society. References [1] A. Jenkins, et al., Evidence for a common binding cavity for three general anesthetics within the GABAA receptor, J. Neurosci. 21 (6) (2001) U7 – U10. [2] I. Belelli, et al., General anaesthetic action at transmitter-gated inhibitory amino acid receptors, Trends Pharmacol. Sci. 20 (12) (1999) 496 – 502. [3] M.I. Banks, R.A. Pearce, Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects, Anesthesiology 90 (1) (1999) 120 – 134. [4] S. Neumahr, et al., Dual action of isoflurane on the gamma-aminobutyric acid (GABA)-mediated currents through recombinant a(1)h(2)g(2L)-GABA(A)-receptor channels, Anesth. Analg. 90 (5) (2000) 1184 – 1190. [5] Q.Y. Liu, et al., Persistent activation of GABAA receptor/Cl( ) channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons, J. Neurophysiol. 84 (3) (2000) 1392 – 1403. [6] D. Bai, et al., Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons, Mol. Pharmacol. 59 (4) (2001) 814 – 824. [7] J.Y. Yeung, et al., Tonically activated GABAA receptors in hippocampal neurons are high-affinity, lowconductance sensors for extracellular GABA, Mol. Pharmacol. 63 (1) (2003) 2 – 8. [8] B.M. Stell, I. Mody, Receptors with different affinities mediate phasic and tonic GABAA conductances in hippocampal neurons, J. Neurosci. (2002). [9] J.M. Fritschy, H. Mohler, GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits, J. Comp. Neurol. 359 (1) (1995) 154 – 194. [10] I. Brunig, et al., Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro, J. Comp. Neurol. 443 (1) (2002) 43 – 55. [11] I. Mody, Distinguishing between GABA(A) receptors responsible for tonic and phasic conductances, Neurochem. Res. 26 (8–9) (2001) 907 – 913. [12] A.L. Scotti, H. Reuter, Synaptic and extrasynaptic g-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development, Proc. Natl. Acad. Sci. U. S. A. 98 (6) (2001) 3489 – 3494. [13] V.B. Caraiscos, et al., Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by a5 subunit-containing g-aminobutyric acid type A receptors, Proc. Natl. Acad. Sci. U. S. A. 101 (10) (March 9) (2004) 3662 – 3667. [14] V.B. Caraiscos, et al., Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane, J. Neurosci. 24 (39) (2004) 8454 – 8458. [15] E.I. Eger, L.J. Saidman, B. Brandstater, Minimum alveolar anesthetic concentration: a standard of anesthetic potency, Anesthesiology 26 (6) (1965) 756 – 763. [16] M. Nakahiro, et al., General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons, FASEB J. 3 (7) (1989) 1850 – 1854. [17] J. Wu, N. Harata, N. Akaike, Potentiation by sevoflurane of the gamma-aminobutyric acid-induced chloride current in acutely dissociated CA1 pyramidal neurones from rat hippocampus, Br. J. Pharmacol. 119 (5) (1996) 1013 – 1021. [18] B. Hille, Classical Mechanisms of Block in Ionic Channels of Excitable Membranes, 3rd ed., Sinauer Associates, Inc., Sunderland, MA, 2001. [19] G. Hapfelmeier, H. Schneck, E. Kochs, Sevoflurane potentiates and blocks GABA-induced currents through recombinant alpha1beta2gamma2 GABAA receptors: implications for an enhanced GABAergic transmission, Eur. J. Anaesthesiol. 18 (6) (2001) 377 – 383.