Dominant-negative suppression of big brain ion channel activity by mutation of a conserved glutamate in the first transmembrane domain.
The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila. The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila.
Yool
Andrea J
AJ
Department of Physiology, BIOS Institute, Arizona Research Labs Division of Neurobiology, University of Arizona, Tucson, AZ 85724, USA. ayool@u.arizona.edu
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1 / Q000737:chemistry / Q000235:genetics; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005799:Genes, Dominant; D018698:Glutamic Acid / Q000235:genetics*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D029721:Drosophila Proteins; D007473:Ion Channels; D008565:Membrane Proteins; C070621:bib protein, Drosophila; D051398:Aquaporin 1; D018698:Glutamic Acid
J Neurosci. 2002 Apr 1;22(7):2530-40
11923418
Biophys J. 1995 Jan;68(1):147-56
7711236
Proc Natl Acad Sci U S A. 2005 Mar 1;102(9):3477-82
15728380
Am J Physiol Renal Physiol. 2003 May;284(5):F893-901
12676734
Structure. 2006 Sep;14(9):1411-23
16962972
Biochemistry. 1996 Jan 16;35(2):538-44
8555225
J Biol Chem. 1994 May 20;269(20):14648-54
7514176
Science. 1996 Aug 30;273(5279):1216-8
8703053
Cell. 1992 May 29;69(5):715-8
1375539
Wilehm Roux Arch Dev Biol. 1983 Mar;192(2):62-74
28305500
Trends Pharmacol Sci. 2002 Dec;23(12):558-62
12457773
Biophys J. 2004 Mar;86(3):1470-8
14990474
Nature. 1999 Nov 11;402(6758):184-7
10647010
Development. 1997 Oct;124(19):3881-93
9367444
Hum Mol Genet. 2000 Sep 22;9(15):2329-34
11001937
J Biol Chem. 1993 Jan 5;268(1):17-20
7677994
Neuropharmacology. 1996;35(7):1007-16
8938731
Physiol Rev. 1999 Oct;79(4):1317-72
10508236
Am J Hum Genet. 2001 Oct;69(4):738-48
11536078
Science. 2002 Apr 19;296(5567):525-30
11964478
News Physiol Sci. 2002 Apr;17:68-72
11909995
Am J Physiol. 1993 Oct;265(4 Pt 2):F463-76
7694481
Curr Eye Res. 1995 Dec;14(12):1095-1100
8974838
Proc Natl Acad Sci U S A. 2005 Feb 8;102(6):2192-7
15671159
Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11110-4
1722319
Nature. 1990 May 10;345(6271):163-7
1692392
Science. 2000 Oct 20;290(5491):481-6
11039922
Development. 1992 Sep;116(1):31-40
1483394
Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):14217-22
16968783
Nature. 2001 Dec 20-27;414(6866):872-8
11780053
Biochemistry. 1999 Jan 19;38(3):923-8
9893987
J Neurosci. 2006 Jul 26;26(30):7811-9
16870726
Crit Rev Biochem Mol Biol. 1993;28(3):235-57
8325040
Mol Pharmacol. 2000 Mar;57(3):576-88
10692499
Hepatology. 2004 Jan;39(1):109-16
14752829
Science. 1992 Aug 28;257(5074):1225-30
1519059