title: Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function.
abstract: The subfamily of G-protein-linked inwardly rectifying potassium channels (GIRKs) is coupled to G-protein receptors throughout the CNS and in the heart. We used mutational analysis to address the role of a specific hydrophobic region of the GIRK1 subunit. Deletion of the GIRK1 C-terminal residues 330-384, as well as the point mutation I331R, resulted in a decrease in channel function when coexpressed with GIRK4 in oocytes and in COS-7 cells. Surface protein expression of GIRK1 I331R coexpressed with GIRK4 was comparable with wild type, indicating that subunits assemble and are correctly localized to the membrane. Subsequent mutation of homologous residues in both the GIRK4 subunit and Kir2.1 (Gbetagamma-independent inward rectifier) also resulted in a decrease in channel function. Intracellular domain associations resulted in the coimmunoprecipitation of the GIRK1 N and C termini and GIRK4 N and C termini. The point mutation I331R in the GIRK1 C terminus or L337R in the GIRK4 C terminus decreased the association between the N and C termini. Mutation of a GIRK1 N-terminal hydrophobic residue, predicted structurally to interact with the C-terminal domain, also resulted in a decrease in channel function and termini association. We hypothesize that the hydrophobic nature of this GIRK1 subunit region is critical for interaction between adjacent termini and is permissive for channel gating. In addition, the homologous mutation in cytoplasmic domains of Kir2.1 (L330R) did not disrupt association, suggesting that the overall structural integrity of this region is critical for inward rectifier function.
authors.affiliation: Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Radmila
authors.initials: R
authors.lastname: Sarac
authors.forename: Ping
authors.initials: P
authors.lastname: Hou
authors.forename: Karen M
authors.initials: KM
authors.lastname: Hurley
authors.forename: David
authors.initials: D
authors.lastname: Hriciste
authors.forename: Noam A
authors.initials: NA
authors.lastname: Cohen
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Nelson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology; D002522:Chlorocebus aethiops; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology; D017136:Ion Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000235:genetics; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C497068:Kcnj3 protein, rat; C489461:Kir2.1 channel; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D002217:Carbachol; D011188:Potassium
references.citation: Neuron. 2003 Apr 24;38(2):225-35
references.pmid: 12718857
references.citation: J Biol Chem. 1995 Jun 16;270(24):14604-10
references.pmid: 7540174
references.citation: Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1307-12
references.pmid: 9448327
references.citation: Nature. 1993 Mar 11;362(6416):127-33
references.pmid: 7680768
references.citation: Annu Rev Neurosci. 1997;20:91-123
references.pmid: 9056709
references.citation: Science. 2003 Jun 20;300(5627):1922-6
references.pmid: 12738871
references.citation: Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6542-6
references.pmid: 7604029
references.citation: Neuron. 1995 Nov;15(5):1133-43
references.pmid: 7576656
references.citation: J Biol Chem. 1999 Nov 19;274(47):33393-7
references.pmid: 10559219
references.citation: J Biol Chem. 1995 Dec 8;270(49):29059-62
references.pmid: 7493925
references.citation: Am J Physiol. 1996 Jul;271(1 Pt 2):H379-85
references.pmid: 8760196
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):76-81
references.pmid: 14681552
references.citation: Eur J Neurosci. 2003 Oct;18(8):2110-8
references.pmid: 14622172
references.citation: Science. 1991 May 24;252(5009):1162-4
references.pmid: 2031185
references.citation: Biochem Biophys Res Commun. 1995 Jul 26;212(3):1022-8
references.pmid: 7626088
references.citation: Nature. 1993 Aug 26;364(6440):802-6
references.pmid: 8355805
references.citation: Cell. 2002 Dec 27;111(7):957-65
references.pmid: 12507423
references.citation: J Biol Chem. 1998 Oct 16;273(42):27499-504
references.pmid: 9765280
references.citation: J Biol Chem. 1995 Dec 1;270(48):28660-7
references.pmid: 7499385
references.citation: FEBS Lett. 1997 Apr 1;405(3):291-8
references.pmid: 9108307
references.citation: FEBS Lett. 2001 Nov 9;508(1):85-9
references.pmid: 11707273
references.citation: J Biol Chem. 1998 Feb 27;273(9):5271-8
references.pmid: 9478984
references.citation: Curr Opin Neurobiol. 2002 Jun;12(3):287-92
references.pmid: 12049935
references.citation: Cell Signal. 1997 Dec;9(8):551-73
references.pmid: 9429760
references.citation: J Biol Chem. 1998 Jul 3;273(27):16946-52
references.pmid: 9642257
references.citation: J Biol Chem. 2000 Nov 17;275(46):36211-6
references.pmid: 10956667
references.citation: Biochem Biophys Res Commun. 1995 Mar 28;208(3):1166-73
references.pmid: 7702616
references.citation: Mol Cell. 2002 Sep;10(3):469-81
references.pmid: 12408817
references.citation: J Biol Chem. 2002 Feb 22;277(8):6088-96
references.pmid: 11741896
references.citation: J Biol Chem. 1999 Apr 30;274(18):12517-24
references.pmid: 10212228
references.citation: Nature. 2002 May 30;417(6888):523-6
references.pmid: 12037560
references.citation: Neuron. 2002 Feb 28;33(5):715-29
references.pmid: 11879649
references.citation: Trends Biochem Sci. 1996 Oct;21(10):375-82
references.pmid: 8918191
references.citation: J Biol Chem. 2004 Mar 19;279(12):11890-7
references.pmid: 14703518
references.citation: Mol Cell Neurosci. 1997;9(3):194-206
references.pmid: 9245502
references.citation: J Biol Chem. 1997 Apr 18;272(16):10823-30
references.pmid: 9099737
references.citation: Nature. 1987 Jan 22-28;325(6102):321-6
references.pmid: 2433589
references.citation: J Biol Chem. 1998 Dec 18;273(51):34575-9
references.pmid: 9852128
references.citation: J Physiol. 1999 Nov 1;520 Pt 3:645-51
references.pmid: 10545132
references.citation: J Biol Chem. 2003 Aug 1;278(31):29174-83
references.pmid: 12743112
references.citation: J Neurosci. 1999 Feb 1;19(3):1006-17
references.pmid: 9920664
references.citation: J Biol Chem. 2000 Oct 13;275(41):31573-80
references.pmid: 10896660
references.citation: Neuron. 2001 Mar;29(3):669-80
references.pmid: 11301026
references.citation: J Biol Chem. 1999 Jan 22;274(4):2571-82
references.pmid: 9891030
references.citation: Synapse. 1989;3(3):234-8
references.pmid: 2541514
references.citation: Neuron. 1996 Feb;16(2):423-9
references.pmid: 8789957
references.citation: Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10235-9
references.pmid: 8234283
references.citation: J Physiol. 2004 Mar 16;555(Pt 3):643-57
references.pmid: 14724209
references.citation: Nature. 1994 Jul 14;370(6485):143-6
references.pmid: 8022483
references.citation: Nature. 1995 Mar 9;374(6518):135-41
references.pmid: 7877685
references.citation: Diabetes. 2002 Dec;51 Suppl 3:S377-80
references.pmid: 12475778
references.citation: J Neurosci. 1999 Oct 1;19(19):8327-36
references.pmid: 10493734
references.citation: FEBS Lett. 1994 Oct 10;353(1):37-42
references.pmid: 7926018
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):8430-5
references.pmid: 12034888
references.citation: J Neurosci. 1996 Jun 1;16(11):3559-70
references.pmid: 8642402