title: Structural order in Pannexin 1 cytoplasmic domains.
abstract: Pannexin 1 forms ion and metabolite permeable hexameric channels with abundant expression in the central nervous system and elsewhere. Although pannexin 1 does not form intercellular channels, a common channel topology and oligomerization state, as well as involvement of the intracellular carboxyl terminal (CT) domain in channel gating, is shared with connexins. In this study, we characterized the secondary structure of the mouse pannexin 1 cytoplasmic domains to complement structural studies of the transmembrane segments and compare with similar domains from connexins. A combination of structural prediction tools and circular dichroism revealed that, unlike connexins (predominately intrinsically disordered), cytosolic regions of pannexin 1 contain approximately 50% secondary structure, a majority being α-helical. Moreover, prediction of transmembrane domains uncovered a potential membrane interacting region (I360-G370) located upstream of the caspase cleavage site (D375-D378) within the pannexin 1 CT domain. The α-helical content of a peptide containing these domains (G357-S384) increased in the presence of detergent micelles providing evidence of membrane association. We also purified a pannexin 1 CT construct containing the caspase cleavage site (M374-C426), assigned the resonances by NMR, and confirmed cleavage by Caspase-3 in vitro. On the basis of these structural studies of the cytoplasmic domains of pannexin 1, we propose a mechanism for the opening of pannexin 1 channels upon apoptosis, involving structural changes within the CT domain.
authors.affiliation: University of Nebraska Medical Center; Department of Biochemistry and Molecular Biology; Omaha, NE USA.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Spagnol
authors.affiliation: University of Nebraska Medical Center; Department of Biochemistry and Molecular Biology; Omaha, NE USA.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Sorgen
authors.affiliation: Albert Einstein College of Medicine; Rose F. Kennedy Center; Bronx, NY USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Spray
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D002942:Circular Dichroism; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D017630:Connexins; D009419:Nerve Tissue Proteins; C503631:Panx1 protein, mouse; D011994:Recombinant Proteins; D053148:Caspase 3
keywords: Caspase-3; NMR; Panx1; circular dichroism; cytoplasmic domains
references.citation: J Biol Chem. 2002 Sep 27;277(39):36706-14
references.pmid: 12151412
references.citation: J Biomol NMR. 1994 Sep;4(5):603-14
references.pmid: 22911360
references.citation: J Cell Biol. 2006 Aug 14;174(4):535-46
references.pmid: 16908669
references.citation: Protein Eng. 2000 Nov;13(11):739-43
references.pmid: 11161104
references.citation: Neuropharmacology. 2013 Dec;75:594-603
references.pmid: 23583931
references.citation: Physiology (Bethesda). 2006 Apr;21:103-14
references.pmid: 16565476
references.citation: J Gen Physiol. 2010 Nov;136(5):515-27
references.pmid: 20937692
references.citation: J Cell Sci. 2007 Nov 1;120(Pt 21):3772-83
references.pmid: 17925379
references.citation: Science. 1999 Feb 19;283(5405):1176-80
references.pmid: 10024245
references.citation: Nature. 2010 Oct 14;467(7317):863-7
references.pmid: 20944749
references.citation: J Cell Biol. 1977 Dec;75(3):769-87
references.pmid: 562887
references.citation: Am J Physiol Cell Physiol. 2008 Sep;295(3):C752-60
references.pmid: 18596211
references.citation: J Cell Biol. 2007 Dec 17;179(6):1301-9
references.pmid: 18086922
references.citation: Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W197-201
references.pmid: 18463136
references.citation: J Mol Biol. 1999 Sep 17;292(2):195-202
references.pmid: 10493868
references.citation: J Biomol NMR. 2010 Mar;46(3):245-50
references.pmid: 20127391
references.citation: J Biol Chem. 2004 Dec 24;279(52):54695-701
references.pmid: 15492000
references.citation: J Mol Biol. 1992 May 20;225(2):487-94
references.pmid: 1593632
references.citation: FEBS Lett. 2004 Aug 13;572(1-3):65-8
references.pmid: 15304325
references.citation: J Mol Biol. 1980 Apr;138(2):149-78
references.pmid: 7411608
references.citation: Protein Eng. 1997 Jun;10(6):673-6
references.pmid: 9278280
references.citation: Eur J Biochem. 1995 Jun 1;230(2):634-49
references.pmid: 7607238
references.citation: Nature. 2002 May 30;417(6888):523-6
references.pmid: 12037560
references.citation: Biochemistry. 1998 Oct 13;37(41):14613-22
references.pmid: 9772190
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: J Biol Chem. 2010 Aug 6;285(32):24420-31
references.pmid: 20516070
references.citation: Pharmacol Rev. 2010 Sep;62(3):405-96
references.pmid: 20716669
references.citation: J Biol Chem. 2012 Mar 30;287(14):11303-11
references.pmid: 22311983
references.citation: Bioorg Med Chem. 1999 Jan;7(1):93-103
references.pmid: 10199660
references.citation: Curr Biol. 2000 Jun 29;10(13):R473-4
references.pmid: 10898987
references.citation: J Biol Chem. 2007 Oct 26;282(43):31733-43
references.pmid: 17715132
references.citation: J Neurosci. 2012 Sep 5;32(36):12579-88
references.pmid: 22956847
references.citation: J Neurosci. 2009 May 27;29(21):7092-7
references.pmid: 19474335
references.citation: Nature. 2007 Dec 13;450(7172):1026-30
references.pmid: 18075582
references.citation: Biochemistry. 1992 Sep 22;31(37):8790-8
references.pmid: 1390666
references.citation: FEBS Lett. 2006 Jan 9;580(1):239-44
references.pmid: 16364313
references.citation: Biopolymers. 2009;92(3):173-82
references.pmid: 19226516
references.citation: Biomol NMR Assign. 2013 Oct;7(2):293-7
references.pmid: 23070843
references.citation: J Biomol NMR. 1994 Mar;4(2):171-80
references.pmid: 8019132
references.citation: J Physiol. 2012 Dec 15;590(24):6257-66
references.pmid: 23070703
references.citation: J Biol Chem. 2007 Feb 23;282(8):5801-13
references.pmid: 17178730
references.citation: J Biomol NMR. 2002 Jul;23(3):245-6
references.pmid: 12238598
references.citation: Circ Res. 2002 Mar 8;90(4):450-7
references.pmid: 11884375
references.citation: J Biol Chem. 2009 Dec 4;284(49):34257-71
references.pmid: 19808665
references.citation: Methods Enzymol. 1996;266:540-53
references.pmid: 8743705
references.citation: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W321-6
references.pmid: 15215403