Inhibition of isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3 by serine 465 sequence-specific peptides.
Excitatory amino acid transporters (EAAT) transport glutamate into cells to regulate glutamate neurotransmission and to maintain nontoxic extracellular glutamate levels for neurons. We showed previously that the commonly used volatile anesthetic isoflurane increases the transporting activity of EAAT3, the major neuronal EAAT. This effect requires a protein kinase C (PKC) α-mediated and S465-dependent EAAT3 redistribution to the plasma membrane. Thus, we hypothesize that specific peptides can be designed to block this effect. We conjugated a 10-amino acid synthetic peptide with a sequence identical to that of EAAT3 around the S465 to a peptide that can facilitate permeation of the plasma membrane. This fusion peptide inhibited the isoflurane-increased EAAT3 activity and redistribution to the plasma membrane in C6 cells and hippocampus. It did not affect the basal EAAT3 activity. This peptide also attenuated isoflurane-induced increase of PKCα in the immunoprecipitates produced by an anti-EAAT3 antibody. A scrambled peptide that has the same amino acid composition as the S465 sequence-specific peptide but has a random sequence did not change the effects of isoflurane on EAAT3. The S465 sequence-specific peptide, but not the scrambled peptide, is a good PKCα substrate in in vitro assay. These peptides did not affect cell viability. These results, along with our previous findings, strongly suggest that PKCα interacts with EAAT3 to regulate its functions. The S465 sequence-specific peptide may interrupt this interaction and is an effective inhibitor for the regulation of EAAT3 activity and trafficking by PKCα and isoflurane.
Department of Anesthesiology, University of Virginia Health System, Charlottesville, VA 22908, USA.
Yueming
Y
Huang
Liaoliao
L
Li
Jacqueline M
JM
Washington
Xuebing
X
Xu
Julianne J
JJ
Sando
Daowei
D
Lin
Zhiyi
Z
Zuo
D000595:Amino Acid Sequence; D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007530:Isoflurane / Q000494:pharmacology*; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D012694:Serine
D000777:Anesthetics; D050580:Excitatory Amino Acid Transporter 3; D009842:Oligopeptides; C493398:Slc1a1 protein, rat; D012694:Serine; D007530:Isoflurane; D051571:Protein Kinase C-alpha
Neuroscience. 1999;93(3):1167-72
10473281
Mol Pharmacol. 1997 Oct;52(4):606-12
9380023
Nature. 1992 Dec 3;360(6403):467-71
1280334
Science. 1999 Sep 3;285(5433):1569-72
10477521
J Neuropathol Exp Neurol. 2006 Oct;65(10):945-52
17021399
Neuron. 1994 Sep;13(3):713-25
7917301
N Engl J Med. 2003 May 22;348(21):2110-24
12761368
Anesthesiology. 2008 Apr;108(4):643-50
18362596
Eur J Pharm Sci. 2010 Aug 11;40(5):404-11
20433920
Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):664-8
8290579
Peptides. 2008 Jun;29(6):904-11
18343535
Nat Neurosci. 2002 Feb;5(2):155-61
11788834
Mol Pharmacol. 1998 Feb;53(2):195-201
9463476
Prog Neurobiol. 2001 Sep;65(1):1-105
11369436
J Neurosci. 1999 Jan 15;19(2):562-9
9880576
J Biol Chem. 1998 Dec 18;273(51):34022-7
9852057
Anesthesiology. 2010 Mar;112(3):546-56
20124985
Biochem J. 1996 Jul 15;317 ( Pt 2):583-8
8713089
J Cereb Blood Flow Metab. 2011 May;31(5):1283-92
21139629
Neuroreport. 2001 Apr 17;12(5):1077-80
11303749
Neuroscience. 2009 Dec 1;164(2):497-506
19679170
J Neurosci. 1998 Apr 1;18(7):2475-85
9502808
Anesthesiology. 2007 Jan;106(1):92-9; discussion 8-10
17197850
Int J Neurosci. 2009;119(9):1419-28
19922365
J Neurosci. 1994 Sep;14(9):5559-69
7521911
Anesth Analg. 2009 Nov;109(5):1506-10
19843789
Biochim Biophys Acta. 1995 May 11;1244(1):233-7
7766664
J Physiol. 1996 Jun 1;493 ( Pt 2):419-23
8782106
Anesthesiology. 2000 May;92(5):1343-9
10781280
J Biol Chem. 2006 Dec 15;281(50):38133-8
17062570
J Biol Chem. 2000 Apr 21;275(16):11993-2002
10766830
Curr Opin Biotechnol. 2002 Feb;13(1):52-6
11849958
Nat Neurosci. 2006 Jan;9(1):119-26
16311588
Biochem J. 1998 Mar 15;330 ( Pt 3):1433-42
9494117
Mol Pharmacol. 2005 May;67(5):1522-33
15709112
J Neurochem. 2007 Dec;103(5):1917-31
17868307
Anesthesiology. 1995 May;82(5):1199-208
7741295
Prog Brain Res. 1998;116:23-43
9932368
Anesthesiology. 2003 Dec;99(6):1346-53
14639147
Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9706-11
10931942
J Neurosci. 2009 Nov 18;29(46):14581-95
19923291
Anesthesiology. 2002 Jun;96(6):1492-7
12170065