Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis.
M-type K(+) channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.
Bal
Manjot
M
Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Zhang
Jie
J
Zaika
Oleg
O
Hernandez
Ciria C
CC
Shapiro
Mark S
MS
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression; D006801:Humans; D051656:KCNQ Potassium Channels / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D018408:Patch-Clamp Techniques / Q000379:methods; D020836:Protein Structure, Quaternary / Q000235:genetics; D051381:Rats
D051656:KCNQ Potassium Channels
Neuron. 1992 Mar;8(3):493-7
1550674
J Neurosci. 2006 Oct 4;26(40):10253-69
17021181
Biophys J. 2008 Dec;95(11):5121-37
18790849
J Physiol. 2008 Feb 1;586(3):795-802
18048450
Hear Res. 2001 Mar;153(1-2):132-45
11223304
J Biol Chem. 2005 Sep 9;280(36):31347-52
16002409
Nat Neurosci. 2003 Jun;6(6):564-71
12754513
J Biol Chem. 2000 Jul 21;275(29):22395-400
10787416
Pharmacol Ther. 2001 Apr;90(1):1-19
11448722
J Physiol. 2006 Aug 15;575(Pt 1):49-67
16777936
J Neurosci. 2003 Jun 15;23(12):5012-9
12832524
Nat Rev Neurosci. 2000 Oct;1(1):21-30
11252765
J Biol Chem. 2003 Apr 4;278(14):11739-45
12529362
J Neurosci. 2000 Mar 1;20(5):1710-21
10684873
Neurology. 2003 Jul 8;61(1):131-4
12847176
Br J Pharmacol. 2000 Feb;129(3):413-5
10711337
Neuron. 2007 Mar 1;53(5):663-75
17329207
Nat Cell Biol. 2005 Nov;7(11):1066-73
16228013
J Physiol. 2006 Aug 15;575(Pt 1):175-89
16777937
J Neurosci. 2003 Jan 1;23(1):84-95
12514204
EMBO Rep. 2003 Jan;4(1):76-81
12524525
J Neurosci. 2004 Jun 2;24(22):5079-90
15175377
J Biol Chem. 2004 Jun 25;279(26):27729-34
15096495
Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16374-8
15528278
J Neurosci. 2002 Jan 15;22(2):RC199
11784811
Nat Rev Neurosci. 2005 Nov;6(11):850-62
16261179
J Neurosci. 2004 Feb 4;24(5):1236-44
14762142
J Physiol. 2008 Jun 15;586(12):2827-40
18440995
J Neurosci. 2005 Oct 26;25(43):9825-35
16251430
Neuron. 2004 May 13;42(3):411-21
15134638
Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4914-9
10781098
Cell Calcium. 2003 May-Jun;33(5-6):463-70
12765691
Methods Enzymol. 2003;361:1-33
12624904
Mol Biol Cell. 2005 Aug;16(8):3538-51
15901836
Science. 1990 Oct 12;250(4978):276-9
2218530
J Neurophysiol. 2006 May;95(5):3105-12
16467425
Methods Enzymol. 2003;360:542-60
12622167
J Physiol. 2008 Mar 1;586(5):1351-63
18187471
J Biol Chem. 2000 May 5;275(18):13343-8
10788442
Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):4333-8
10760300
Physiology (Bethesda). 2005 Dec;20:408-16
16287990
Neuron. 1992 Mar;8(3):483-91
1550673
Br J Pharmacol. 2001 Jan;132(2):381-4
11159685
Am J Med Genet. 2001 Summer;106(2):146-59
11579435
Biophys J. 1998 May;74(5):2702-13
9591694
J Neurosci. 2006 Nov 22;26(47):12325-38
17122058
J Physiol. 2007 Aug 1;582(Pt 3):911-6
17412763
J Physiol. 2006 Oct 1;576(Pt 1):235-56
16840518
Arch Neurol. 2003 Apr;60(4):496-500
12707061
J Cell Biol. 2003 Mar 3;160(5):629-33
12615908
J Neurosci. 2007 Aug 15;27(33):8914-26
17699673
Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6160-5
15827117
Br J Pharmacol. 2002 Dec;137(8):1173-86
12466226
Nat Rev Mol Cell Biol. 2001 Apr;2(4):268-75
11283724
J Neurosci. 2003 Aug 6;23(18):7227-36
12904483
Science. 1998 Dec 4;282(5395):1890-3
9836639
J Neurosci. 2004 Oct 13;24(41):9146-52
15483133
Science. 1998 Jan 16;279(5349):403-6
9430594
Neuroscience. 2003;120(2):353-64
12890507
J Neurophysiol. 2007 Jan;97(1):283-95
17050829
J Neurosci. 2004 Jan 21;24(3):592-7
14736843
J Neurophysiol. 2006 Jun;95(6):3480-95
16495357
J Physiol. 2008 Apr 1;586(7):1785-9
18174212
J Physiol. 2007 Apr 1;580(Pt 1):51-65
17185339
J Neurosci. 2001 Dec 15;21(24):9529-40
11739564
J Physiol. 2005 Aug 1;566(Pt 3):689-715
15890705
J Biol Chem. 2000 Aug 4;275(31):24089-95
10816588
J Physiol. 2002 Oct 1;544(Pt 1):29-37
12356878
Neuron. 2002 Dec 5;36(5):891-6
12467592
Cell. 1999 Feb 5;96(3):437-46
10025409
J Physiol. 2008 Apr 1;586(7):1791-801
18238816
J Biol Chem. 1998 Jul 31;273(31):19419-23
9677360
Cell Physiol Biochem. 2006;18(1-3):57-66
16914890
J Neurosci. 2006 Apr 5;26(14):3757-66
16597729
Nature. 1998 Dec 17;396(6712):687-90
9872318
Handb Exp Pharmacol. 2006;(171):221-33
16610346
J Pharmacol Toxicol Methods. 2005 May-Jun;51(3):177-85
15862463
J Physiol. 2008 May 1;586(9):2307-20
18339689
Neuron. 1992 Nov;9(5):861-71
1419000
Methods Mol Biol. 2006;337:167-83
16929947
J Neurosci. 2005 Aug 10;25(32):7449-58
16093396