Protein interactions involving the gamma2 large cytoplasmic loop of GABA(A) receptors modulate conductance.
Native GABA(A) channels display a single-channel conductance ranging between approximately 10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane, suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the gamma2 subunit (gamma(381-403)) of the GABA(A) receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABA(A) receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (gamma(386-403)) known to compete with the gamma2-GABARAP interaction did not affect the conductance of recombinant alphabetagamma receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the gamma2 MA helix is able to self-associate support a mechanism whereby adjacent GABA(A) receptors interact via their gamma2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.
Everitt
Andrea B
AB
Structural Biology Programme, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
Seymour
Victoria A L
VA
Curmi
John
J
Laver
Derek R
DR
Gage
Peter W
PW
Tierney
M Louise
ML
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain; D003975:Diazepam / Q000494:pharmacology; D004594:Electrophysiology; D005786:Gene Expression Regulation / Q000502:physiology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
C502186:GABARAP protein, rat; C546280:Gabrg2 protein, mouse; C546281:Gabrg2 protein, rat; D008869:Microtubule-Associated Proteins; D011963:Receptors, GABA-A; D003975:Diazepam
Nature. 1999 Jan 7;397(6714):69-72
9892355
J Membr Biol. 1993 Dec;136(3):273-80
8114077
J Neurosci. 1988 Jan;8(1):289-95
2448430
Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11557-62
10984509
J Membr Biol. 2005 May;205(1):17-28
16245039
Biophys J. 1987 Feb;51(2):255-63
2435330
J Biol Chem. 2004 Feb 20;279(8):6967-75
14660627
J Neurosci. 2002 Feb 1;22(3):684-97
11826098
Nature. 1997 Jul 3;388(6637):71-5
9214504
J Biol Chem. 2005 Jul 22;280(29):27062-8
15927954
J Biol Chem. 2009 Jan 23;284(4):2023-30
19049967
J Biol Chem. 2006 Nov 24;281(47):35699-708
16954214
Annu Rev Neurosci. 1994;17:569-602
7516126
Mol Cell Neurosci. 2006 Jan;31(1):1-14
16181787
J Steroid Biochem Mol Biol. 2007 Apr;104(1-2):35-44
17254775
J Biol Chem. 2004 May 21;279(21):21701-6
15007071
J Neurochem. 2002 Dec;83(5):1164-71
12437587
Biochemistry. 2000 Aug 29;39(34):10347-52
10956024
J Neurosci. 1998 Mar 1;18(5):1693-703
9464994
J Membr Biol. 2009 Feb;227(3):111-22
19153634
Nature. 2000 Jul 27;406(6794):405-10
10935636
Nature. 2001 Feb 22;409(6823):1047-51
11234014
J Mol Biol. 2005 Mar 4;346(4):967-89
15701510
J Neurosci. 1993 Apr;13(4):1429-40
7681870
Eur J Pharmacol. 2003 May 9;468(2):75-82
12742514
Nature. 2000 Jan 20;403(6767):274-80
10659839
J Neurosci. 2007 Dec 26;27(52):14326-37
18160640
Science. 1998 Dec 18;282(5397):2220-6
9856938
J Neurochem. 2002 Mar;80(5):815-23
11948245
J Physiol. 1999 May 1;516 ( Pt 3):719-37
10200421
Mol Pharmacol. 2000 Sep;58(3):463-9
10953037
Structure. 2001 Mar 7;9(3):215-20
11286888
Proc Biol Sci. 1994 Feb 22;255(1343):167-72
8165229
Nature. 2003 Jul 17;424(6946):321-4
12867984
Proc Biol Sci. 1992 Nov 23;250(1328):99-105
1282261
J Biol Chem. 2004 Dec 10;279(50):52517-25
15456793
J Biol Chem. 2006 Mar 24;281(12):8062-71
16407231
J Neurosci. 2007 Jun 20;27(25):6788-99
17581966
J Physiol. 1994 Feb 15;475(1):69-82
7514665
Int J Biochem Cell Biol. 2008;40(5):968-79
18083058
J Neurophysiol. 1985 Jul;54(1):134-42
2411883
J Neurosci. 2008 Feb 6;28(6):1421-6
18256262
EMBO J. 2002 May 1;21(9):2087-94
11980705
J Neurosci. 2003 Feb 15;23(4):1246-53
12598613