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TRESK is a mammalian anesthetic-sensitive K channel
International Congress Series 1283 (2005) 181 – 184
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TRESK is a mammalian anesthetic-sensitive K channel C. Spencer Yost * Department of Anesthesia and Perioperative Care, University of California, 513 Parnassus Avenue, Room S-261, Box 0542, San Francisco, CA 94143-0542, USA
Abstract. Several members of the two-pore potassium channel family (K2P) display currents that are potentiated by clinical concentrations of volatile anesthetics. Fifteen human family members are known at present. In this presentation we show data on the anesthetic sensitivity of the most recently isolated K2P channel member, TRESK. Human TRESK is strongly potentiated by volatile anesthetics at near sub-clinical concentrations. Rodent (mouse and rat) TRESKs are also potentiated but with significantly lower efficacy. Species-specific differences are also found between rodent and human TRESKs in extracellular pH and zinc sensitivity. TRESK was initially described as having an expression pattern restricted to spinal cord and testis only; however, we have demonstrated TRESK expression by RT-PCR in human and rodent brain and by immunohistochemistry in rat brain. These data identify TRESK as a likely candidate for the action of volatile anesthetics, adding to the established role of K2P channels in anesthesia mechanisms. D 2005 Elsevier B.V. All rights reserved. Keywords: Volatile anesthetics; Mechanism; Background potassium channels; Two-pore; TRESK
1. Introduction The mechanism by which the volatile anesthetics produce a state of unresponsiveness to painful stimuli remains an important unsolved problem in human biology. Ion channels that reside in the extracellular membrane of excitable cells have been the focus of investigation for at least the last 20 years [1]. Members of the ligand-gated ion channel superfamily, especially those mediating inhibitory cellular influences such as the glycine and GABAA receptors, have garnered the most attention. Recent work has identified a
* Tel.: +1 415 476 5200; fax: +1 415 476 8841. E-mail address: [email protected]. 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.07.045
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class of ion channels called background potassium channels that have also emerged as candidate targets for volatile anesthetics [2]. A specific family of K channels, known for their unique two-pore structure (K2P channel family), is the molecular effector of background K currents [3]; members of this family are strongly potentiated by volatile anesthetics. TRESK (acronym for TWIK-RElated Spinal cord K channel) is the newest member of the K2P family and appears to be intensely activated by clinical concentrations of volatile anesthetics. In this manuscript I report our most recent findings regarding the effect of anesthetics on TRESK and describe its pattern of expression within the central nervous system. These results lend support to an emerging theory, termed elsewhere in this meeting as the bK channel hypothesis of volatile anesthetic actionQ. 2. Materials and methods Pharmacologic studies were performed by superfusion of agents onto Xenopus laevis oocytes expressing TRESK. cRNA transcripts were synthesized from linearized cDNA template of TRESK using T3 RNA polymerases (mMessage mMachine; Ambion, Austin, TX). Defolliculated oocytes were injected with 1–15 ng cRNA using standard methods for oocyte preparation and maintenance. One to four days after injection, two-electrode voltage-clamp recordings were performed at room temperature (GeneClamp 500B; Axon Instruments, Foster City, CA). Voltage pulse protocols were applied from a holding potential of 60 mV using 1 s voltage pulse steps ranging from 120 to + 60 mV in 20 mV increments, with 1.5-s interpulse intervals. All two-electrode voltage clamp experiments were performed using frog Ringer’s solution (bFRQ, composition in mM: 115 NaCl, 5 KCl, 1.8 CaCl2, 10 HEPES, pH 7.6). Recordings were obtained in a 25 Al recording chamber at flow rates of 1–4 ml/min. Signals were filtered using a low-pass filter set at a 50–100 Hz cutoff prior to sampling at 100–1000 Hz. Water-injected oocytes were used as controls. Immunohistochemistry was performed using a polyclonal antisera made by injecting rabbits with a favorable decapeptide sequence derived from the intracellular aminoterminus portion of TRESK. Immunostaining of 40 Am rat brain sections with anti-TASK3 diluted 1:500 was done overnight at room temperature. Negative controls were incubated with pre-immune rabbit IgG alone and with the antibodies preabsorbed for 1 h with the
Fig. 1. Baseline currents recorded from human TRESK expressing oocyte exposed to isoflurane. Membrane potential was stepped to positive test potentials under two-electrode voltage clamp.
C.S. Yost / International Congress Series 1283 (2005) 181–184
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immunizing peptide (1:1 mg/mg ratio). Sections were incubated with a secondary antibody peroxidase complex for 1 h at room temperature, and a nickel-enhanced immunodetection with diaminobenzidine (DAB) was performed. Sections were photographed by light microscopy using a Nikon Eclipse E800 microscope with a Nikon DXM-1200 digital camera system (Nikon Inc., Instrument Group, Melville, NY.
3. Results Injection of cRNA transcribed from human, mouse and rat TRESK cDNAs produced robust baseline currents (1–8 AA of outward current at positive pulses (Fig. 1), control). Water-injected oocytes displayed negligible baseline currents (b 100 nA). Superfusion of the volatile anesthetic isoflurane at an aqueous concentration of 1.3–1.5 MAC (400 AM) produced intense potentiation of outward human TRESK currents (Fig. 1, middle). Strong potentiation of human TRESK currents was observed at concentrations as low as 50 AM (b 0.2 MAC). In contrast, rat and mouse TRESK currents were also potentiated by volatile anesthetics but at about four- to
Fig. 2. Immunolocalization of TRESK using rabbit polyclonal antisera. Staining was performed on 50 Am coronal cryosections of rat brain using Envision secondary anti-rabbit IgG-HRP polymer (Dako) and diaminobenzidine staining kit (Vector). OB = olfactory bulb, Ctx = cortex, Cer = cerebellum, Hp = hippocampus, LV = lateral ventricle, SVZ = subventricular zone, LSN = lateral septal nucleus. Moving clockwise from upper left: cortical motor neurons in all layers are strongly positive, including cell bodies, and neurites (A). Similar staining was also seen in frontal, sensory and occipital cortex. In the cerebellum (B), Purkinje cells are strongly positive with weaker staining of granular and basket neurons. Preimmune serum produced no staining. In the pontine reticular nucleus (C) neurons are strongly positive. Within the hippocampus (D), finally, subventricular zone and lateral septal nucleus neurons strongly positive in cell bodies and neuropil.
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sixfold lower efficacy—instead of a maximal potentiation of 2–300% that was shown for human TRESK the maximal potentiation of rodent TRESK was ~ 50%. To determine the pattern of expression of TRESK within the mammalian CNS experiments were carried out to determine the level of transcript (RT-PCR) and protein (immunohistochemistry) in regions of spinal cord and brain. TRESK transcript could be amplified from human brain and spinal cord but not from heart total RNA [4]. A distinct pattern of protein expression in areas of rat brain could be discerned using specific polyclonal antisera. As shown in Fig. 2, TRESK protein expression could be detected in cortex, cerebellum, pons, hippocampus and septum. As controls, incubation with preimmune sera or pre-absorption of the anti-TRESK antisera with immunizing peptide completely eliminated staining.
4. Discussion and conclusions In this report we demonstrate that currents passed by the most recently isolated K2P channel, TRESK, when expressed heterologously, are strongly potentiated by volatile anesthetics in the clinically used concentration range. This pattern of response is similar to that found before for other members of the K2P channel family. However, no other K2P channel has shown the degree of sensitivity to volatile anesthetics that we have shown here for TRESK. For example, 1 mM halothane and 2 mM isoflurane potentiate TREK-1 (KCNK2) and TASK-1 (KCNK3) in the range of 20–60% [5]; TRESK currents were increased by 150 to more than 300% at lower concentrations. Furthermore, significant potentiation was found at concentrations below 100 AM, aqueous concentrations, which in clinical practice would be considered blightQ or sub-surgical levels. Because TRESK has been identified in spinal cord tissue we propose that TRESK channels play an important role in causing immobility to surgical stimulation produced by volatile anesthetics. In addition, by identifying TRESK transcript in brain RNA and by immunohistochemistry in rat brain, a role for TRESK in mediating other components of general anesthesia is also feasible. Acknowledgments This work was supported by grant GM58149 from the National Institute of General Medical Sciences. References [1] N.P. Franks, W.R. Lieb, Molecular and cellular mechanisms of general anaesthesia, Nature 367 (1994) 607 – 614. [2] N.P. Franks, W.R. Lieb, Background K+ channels: an important target for volatile anesthetics?, Nat. Neurosci. 2 (1999) 395 – 396. [3] S.A. Goldstein, et al., Potassium leak channels and the KCNK family of two-P-domain subunits, Nat. Rev., Neurosci. 2 (2001) 175 – 184. [4] C. Liu, et al., Potent activation of the 2P K channel TRESK by clinical concentrations of volatile anesthetics, Anesth. Analg. 99 (2004) 1715 – 1722. [5] A.J. Patel, et al., Inhalational anesthetics activate two-pore-domain background K+ channels, Nat. Neurosci. 2 (1999) 422 – 426.
www.ics-elsevier.com
TRESK is a mammalian anesthetic-sensitive K channel C. Spencer Yost * Department of Anesthesia and Perioperative Care, University of California, 513 Parnassus Avenue, Room S-261, Box 0542, San Francisco, CA 94143-0542, USA
Abstract. Several members of the two-pore potassium channel family (K2P) display currents that are potentiated by clinical concentrations of volatile anesthetics. Fifteen human family members are known at present. In this presentation we show data on the anesthetic sensitivity of the most recently isolated K2P channel member, TRESK. Human TRESK is strongly potentiated by volatile anesthetics at near sub-clinical concentrations. Rodent (mouse and rat) TRESKs are also potentiated but with significantly lower efficacy. Species-specific differences are also found between rodent and human TRESKs in extracellular pH and zinc sensitivity. TRESK was initially described as having an expression pattern restricted to spinal cord and testis only; however, we have demonstrated TRESK expression by RT-PCR in human and rodent brain and by immunohistochemistry in rat brain. These data identify TRESK as a likely candidate for the action of volatile anesthetics, adding to the established role of K2P channels in anesthesia mechanisms. D 2005 Elsevier B.V. All rights reserved. Keywords: Volatile anesthetics; Mechanism; Background potassium channels; Two-pore; TRESK
1. Introduction The mechanism by which the volatile anesthetics produce a state of unresponsiveness to painful stimuli remains an important unsolved problem in human biology. Ion channels that reside in the extracellular membrane of excitable cells have been the focus of investigation for at least the last 20 years [1]. Members of the ligand-gated ion channel superfamily, especially those mediating inhibitory cellular influences such as the glycine and GABAA receptors, have garnered the most attention. Recent work has identified a
* Tel.: +1 415 476 5200; fax: +1 415 476 8841. E-mail address: [email protected]. 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.07.045
182
C.S. Yost / International Congress Series 1283 (2005) 181–184
class of ion channels called background potassium channels that have also emerged as candidate targets for volatile anesthetics [2]. A specific family of K channels, known for their unique two-pore structure (K2P channel family), is the molecular effector of background K currents [3]; members of this family are strongly potentiated by volatile anesthetics. TRESK (acronym for TWIK-RElated Spinal cord K channel) is the newest member of the K2P family and appears to be intensely activated by clinical concentrations of volatile anesthetics. In this manuscript I report our most recent findings regarding the effect of anesthetics on TRESK and describe its pattern of expression within the central nervous system. These results lend support to an emerging theory, termed elsewhere in this meeting as the bK channel hypothesis of volatile anesthetic actionQ. 2. Materials and methods Pharmacologic studies were performed by superfusion of agents onto Xenopus laevis oocytes expressing TRESK. cRNA transcripts were synthesized from linearized cDNA template of TRESK using T3 RNA polymerases (mMessage mMachine; Ambion, Austin, TX). Defolliculated oocytes were injected with 1–15 ng cRNA using standard methods for oocyte preparation and maintenance. One to four days after injection, two-electrode voltage-clamp recordings were performed at room temperature (GeneClamp 500B; Axon Instruments, Foster City, CA). Voltage pulse protocols were applied from a holding potential of 60 mV using 1 s voltage pulse steps ranging from 120 to + 60 mV in 20 mV increments, with 1.5-s interpulse intervals. All two-electrode voltage clamp experiments were performed using frog Ringer’s solution (bFRQ, composition in mM: 115 NaCl, 5 KCl, 1.8 CaCl2, 10 HEPES, pH 7.6). Recordings were obtained in a 25 Al recording chamber at flow rates of 1–4 ml/min. Signals were filtered using a low-pass filter set at a 50–100 Hz cutoff prior to sampling at 100–1000 Hz. Water-injected oocytes were used as controls. Immunohistochemistry was performed using a polyclonal antisera made by injecting rabbits with a favorable decapeptide sequence derived from the intracellular aminoterminus portion of TRESK. Immunostaining of 40 Am rat brain sections with anti-TASK3 diluted 1:500 was done overnight at room temperature. Negative controls were incubated with pre-immune rabbit IgG alone and with the antibodies preabsorbed for 1 h with the
Fig. 1. Baseline currents recorded from human TRESK expressing oocyte exposed to isoflurane. Membrane potential was stepped to positive test potentials under two-electrode voltage clamp.
C.S. Yost / International Congress Series 1283 (2005) 181–184
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immunizing peptide (1:1 mg/mg ratio). Sections were incubated with a secondary antibody peroxidase complex for 1 h at room temperature, and a nickel-enhanced immunodetection with diaminobenzidine (DAB) was performed. Sections were photographed by light microscopy using a Nikon Eclipse E800 microscope with a Nikon DXM-1200 digital camera system (Nikon Inc., Instrument Group, Melville, NY.
3. Results Injection of cRNA transcribed from human, mouse and rat TRESK cDNAs produced robust baseline currents (1–8 AA of outward current at positive pulses (Fig. 1), control). Water-injected oocytes displayed negligible baseline currents (b 100 nA). Superfusion of the volatile anesthetic isoflurane at an aqueous concentration of 1.3–1.5 MAC (400 AM) produced intense potentiation of outward human TRESK currents (Fig. 1, middle). Strong potentiation of human TRESK currents was observed at concentrations as low as 50 AM (b 0.2 MAC). In contrast, rat and mouse TRESK currents were also potentiated by volatile anesthetics but at about four- to
Fig. 2. Immunolocalization of TRESK using rabbit polyclonal antisera. Staining was performed on 50 Am coronal cryosections of rat brain using Envision secondary anti-rabbit IgG-HRP polymer (Dako) and diaminobenzidine staining kit (Vector). OB = olfactory bulb, Ctx = cortex, Cer = cerebellum, Hp = hippocampus, LV = lateral ventricle, SVZ = subventricular zone, LSN = lateral septal nucleus. Moving clockwise from upper left: cortical motor neurons in all layers are strongly positive, including cell bodies, and neurites (A). Similar staining was also seen in frontal, sensory and occipital cortex. In the cerebellum (B), Purkinje cells are strongly positive with weaker staining of granular and basket neurons. Preimmune serum produced no staining. In the pontine reticular nucleus (C) neurons are strongly positive. Within the hippocampus (D), finally, subventricular zone and lateral septal nucleus neurons strongly positive in cell bodies and neuropil.
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C.S. Yost / International Congress Series 1283 (2005) 181–184
sixfold lower efficacy—instead of a maximal potentiation of 2–300% that was shown for human TRESK the maximal potentiation of rodent TRESK was ~ 50%. To determine the pattern of expression of TRESK within the mammalian CNS experiments were carried out to determine the level of transcript (RT-PCR) and protein (immunohistochemistry) in regions of spinal cord and brain. TRESK transcript could be amplified from human brain and spinal cord but not from heart total RNA [4]. A distinct pattern of protein expression in areas of rat brain could be discerned using specific polyclonal antisera. As shown in Fig. 2, TRESK protein expression could be detected in cortex, cerebellum, pons, hippocampus and septum. As controls, incubation with preimmune sera or pre-absorption of the anti-TRESK antisera with immunizing peptide completely eliminated staining.
4. Discussion and conclusions In this report we demonstrate that currents passed by the most recently isolated K2P channel, TRESK, when expressed heterologously, are strongly potentiated by volatile anesthetics in the clinically used concentration range. This pattern of response is similar to that found before for other members of the K2P channel family. However, no other K2P channel has shown the degree of sensitivity to volatile anesthetics that we have shown here for TRESK. For example, 1 mM halothane and 2 mM isoflurane potentiate TREK-1 (KCNK2) and TASK-1 (KCNK3) in the range of 20–60% [5]; TRESK currents were increased by 150 to more than 300% at lower concentrations. Furthermore, significant potentiation was found at concentrations below 100 AM, aqueous concentrations, which in clinical practice would be considered blightQ or sub-surgical levels. Because TRESK has been identified in spinal cord tissue we propose that TRESK channels play an important role in causing immobility to surgical stimulation produced by volatile anesthetics. In addition, by identifying TRESK transcript in brain RNA and by immunohistochemistry in rat brain, a role for TRESK in mediating other components of general anesthesia is also feasible. Acknowledgments This work was supported by grant GM58149 from the National Institute of General Medical Sciences. References [1] N.P. Franks, W.R. Lieb, Molecular and cellular mechanisms of general anaesthesia, Nature 367 (1994) 607 – 614. [2] N.P. Franks, W.R. Lieb, Background K+ channels: an important target for volatile anesthetics?, Nat. Neurosci. 2 (1999) 395 – 396. [3] S.A. Goldstein, et al., Potassium leak channels and the KCNK family of two-P-domain subunits, Nat. Rev., Neurosci. 2 (2001) 175 – 184. [4] C. Liu, et al., Potent activation of the 2P K channel TRESK by clinical concentrations of volatile anesthetics, Anesth. Analg. 99 (2004) 1715 – 1722. [5] A.J. Patel, et al., Inhalational anesthetics activate two-pore-domain background K+ channels, Nat. Neurosci. 2 (1999) 422 – 426.