- Email: [email protected]
Drosophila A-kinase anchor protein 200, protein kinase A and diethylether anesthesia
International Congress Series 1283 (2005) 263 – 264
www.ics-elsevier.com
Drosophila A-kinase anchor protein 200, protein kinase A and diethylether anesthesia Akihiro Tamai a,*, Yoshiharu Tanaka b, Sumiko Gamo b a
Division of Environmental Studies, Graduate School of Frontier Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan b Department of Earth and Life Sciences, Osaka Prefecture University, Osaka, Japan
Abstract. To identify the cause of resistance to diethylether anesthesia in Drosophila A-kinase anchor protein 200 (DAKAP200) mutant, Eth AR 206, we tested genetic interaction with pka-RII EP2162 . The double mutant analysis suggested the existence of genetic interaction between them. Anesthetic concentration of strains with altered endogenous protein kinase A (PKA) activity also showed altered sensitivity to diethylether. This suggests that DAKAP200 mutation altered ether sensitivity by affecting PKA signaling pathway. We speculate that DAKAP200 is involved in phosphorylation regulation of membrane proteins. Many ion channels are known to be directly regulated their conductivity by PKA phosphorylation in mammals and most homologs of those of Drosophila have conserved sequence for PKA phosphorylation site. These channels are considered to be main target molecules of anesthetics at the same time. Our proposal is that DAKAP200 protein affects sensitivity to anesthetics through phosphorylation regulation of ion channels. D 2005 Elsevier B.V. All rights reserved. Keywords: Drosophila A-kinase anchor protein 200; PKA; Genetic interaction
1. Introduction We have identified Eth AR 206 of Drosophila melanogaster as a resistant strain to diethylether anesthesia previously which has a mutation in the locus of Drosophila Akinase anchor protein 200 (DAKAP200) [1]. DAKAP200 binds to protein kinase A regulatory subunit type II (PKA-RII) to localize PKA heterotetramer to plasma membrane [2,3] and expressed in glia of larva [4]. * Corresponding author. E-mail address: [email protected] (A. Tamai). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.038
264
A. Tamai et al. / International Congress Series 1283 (2005) 263–264
Table 1 EC50 (%atm) to diethylether anesthesia in Drosophila strains Strain
Female
Male
Canton S Eth AR 206 pka-RII EP2162 dnc 2 rut 1 amn Eth AR 206; pka-RII EP2162
1.91 F 0.06 2.98 F 0.15**** 1.49 F 0.04**** 2.28 F 0.16 1.75 F 0.05* 1.74 F 0.04* 2.03 F 0.07
1.92 F 0.07 2.71 F 0.13**** 1.41 F 0.04**** 2.37 F 0.15* 1.73 F 0.05* 1.74 F 0.04* 1.69 F 0.06***
*p b 0.05, **p b 0.01, ***p b 0.001, ****p b 0.0005 compared with Canton S.
2. Material and methods We used Eth AR 206, pka-RII EP2162 , dunce (dnc 2 ), rutabaga (rut 1 ) and amnesiac (amn). Double mutant strain Eth AR 206; pka-RII EP2162 was generated by crossing. Canton S was used as wild type. Anesthetic assay was performed as previously described method to estimate ED50 (%atm) and 95% confidence limits [5]. 3. Result Expression level of DAKAP200 is decreased moderately in Eth AR 206 (data not shown). A mutant strain of pka-RII, pka-RII EP2162 , was hypersensitive to ether. Double mutant analysis suggested that pka-RII is on upstream of DAKAP200 as genetic interaction. Then we tested anesthetic concentration of mutant strains of PKA activity on the hypothesis that PKA signaling pathway is involved to sensitivity to ether (Table 1). dnc 2 , which has higher PKA activity, was resistant and vice versa on rut 1 and amn. This suggests that sensitivity to diethylether tends to go along with PKA activity.
4. Discussion Each AKAPs associate PKA with their substrates in a specific manner and reduction of DAKAP200 seems to result in PKA activity reduction near plasma membrane which could make the strain sensitive. A possible explanation on this is that DAKAP200 is specifically involved in activation of component on plasma membrane related to sensitive phenotype, or inactivation of component related to resistance through PKA. An example of former is GABAA receptor and of the latter is voltage-dependent potassium ion channel. They exist both in mammal and in Drosophila, are expected to be major target of anesthetics, and they conserve PKA phosphorylation site in intracellular part and activated/inactivated by PKA. Thus, reduction of DAKAP200 may result in less activated/inactivated component of sensitive/resistant phenotype, and make this strain resistant. References [1] S. Gamo, et al., in: B.W. Urban, M. Barann (Eds.), Molecular and Basic Mechanisms of Anesthesia, Pabst Science Publishers, Lengerich, 2002, pp. 493 – 499. [2] Z. Li, et al., J. Biol. Chem. 274 (38) (1999) 27191 – 27200. [3] E.A. Rossi, et al., J. Biol. Chem. 274 (38) (1999) 27201 – 27210. [4] M.R. Freeman, et al., Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function, Neuron 38 (2003) 567 – 580. [5] S. Gamo, et al., Opposing effects between 60Co gamma-radiation damage and ether anesthesia in anestheticresistant strain of Drosophila melanogaster: evidences in chromosomal analysis, Jpn. J. Genet. 61 (1986) 315 – 328.
www.ics-elsevier.com
Drosophila A-kinase anchor protein 200, protein kinase A and diethylether anesthesia Akihiro Tamai a,*, Yoshiharu Tanaka b, Sumiko Gamo b a
Division of Environmental Studies, Graduate School of Frontier Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan b Department of Earth and Life Sciences, Osaka Prefecture University, Osaka, Japan
Abstract. To identify the cause of resistance to diethylether anesthesia in Drosophila A-kinase anchor protein 200 (DAKAP200) mutant, Eth AR 206, we tested genetic interaction with pka-RII EP2162 . The double mutant analysis suggested the existence of genetic interaction between them. Anesthetic concentration of strains with altered endogenous protein kinase A (PKA) activity also showed altered sensitivity to diethylether. This suggests that DAKAP200 mutation altered ether sensitivity by affecting PKA signaling pathway. We speculate that DAKAP200 is involved in phosphorylation regulation of membrane proteins. Many ion channels are known to be directly regulated their conductivity by PKA phosphorylation in mammals and most homologs of those of Drosophila have conserved sequence for PKA phosphorylation site. These channels are considered to be main target molecules of anesthetics at the same time. Our proposal is that DAKAP200 protein affects sensitivity to anesthetics through phosphorylation regulation of ion channels. D 2005 Elsevier B.V. All rights reserved. Keywords: Drosophila A-kinase anchor protein 200; PKA; Genetic interaction
1. Introduction We have identified Eth AR 206 of Drosophila melanogaster as a resistant strain to diethylether anesthesia previously which has a mutation in the locus of Drosophila Akinase anchor protein 200 (DAKAP200) [1]. DAKAP200 binds to protein kinase A regulatory subunit type II (PKA-RII) to localize PKA heterotetramer to plasma membrane [2,3] and expressed in glia of larva [4]. * Corresponding author. E-mail address: [email protected] (A. Tamai). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.038
264
A. Tamai et al. / International Congress Series 1283 (2005) 263–264
Table 1 EC50 (%atm) to diethylether anesthesia in Drosophila strains Strain
Female
Male
Canton S Eth AR 206 pka-RII EP2162 dnc 2 rut 1 amn Eth AR 206; pka-RII EP2162
1.91 F 0.06 2.98 F 0.15**** 1.49 F 0.04**** 2.28 F 0.16 1.75 F 0.05* 1.74 F 0.04* 2.03 F 0.07
1.92 F 0.07 2.71 F 0.13**** 1.41 F 0.04**** 2.37 F 0.15* 1.73 F 0.05* 1.74 F 0.04* 1.69 F 0.06***
*p b 0.05, **p b 0.01, ***p b 0.001, ****p b 0.0005 compared with Canton S.
2. Material and methods We used Eth AR 206, pka-RII EP2162 , dunce (dnc 2 ), rutabaga (rut 1 ) and amnesiac (amn). Double mutant strain Eth AR 206; pka-RII EP2162 was generated by crossing. Canton S was used as wild type. Anesthetic assay was performed as previously described method to estimate ED50 (%atm) and 95% confidence limits [5]. 3. Result Expression level of DAKAP200 is decreased moderately in Eth AR 206 (data not shown). A mutant strain of pka-RII, pka-RII EP2162 , was hypersensitive to ether. Double mutant analysis suggested that pka-RII is on upstream of DAKAP200 as genetic interaction. Then we tested anesthetic concentration of mutant strains of PKA activity on the hypothesis that PKA signaling pathway is involved to sensitivity to ether (Table 1). dnc 2 , which has higher PKA activity, was resistant and vice versa on rut 1 and amn. This suggests that sensitivity to diethylether tends to go along with PKA activity.
4. Discussion Each AKAPs associate PKA with their substrates in a specific manner and reduction of DAKAP200 seems to result in PKA activity reduction near plasma membrane which could make the strain sensitive. A possible explanation on this is that DAKAP200 is specifically involved in activation of component on plasma membrane related to sensitive phenotype, or inactivation of component related to resistance through PKA. An example of former is GABAA receptor and of the latter is voltage-dependent potassium ion channel. They exist both in mammal and in Drosophila, are expected to be major target of anesthetics, and they conserve PKA phosphorylation site in intracellular part and activated/inactivated by PKA. Thus, reduction of DAKAP200 may result in less activated/inactivated component of sensitive/resistant phenotype, and make this strain resistant. References [1] S. Gamo, et al., in: B.W. Urban, M. Barann (Eds.), Molecular and Basic Mechanisms of Anesthesia, Pabst Science Publishers, Lengerich, 2002, pp. 493 – 499. [2] Z. Li, et al., J. Biol. Chem. 274 (38) (1999) 27191 – 27200. [3] E.A. Rossi, et al., J. Biol. Chem. 274 (38) (1999) 27201 – 27210. [4] M.R. Freeman, et al., Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function, Neuron 38 (2003) 567 – 580. [5] S. Gamo, et al., Opposing effects between 60Co gamma-radiation damage and ether anesthesia in anestheticresistant strain of Drosophila melanogaster: evidences in chromosomal analysis, Jpn. J. Genet. 61 (1986) 315 – 328.