Molecular mechanisms of Slo2 K+channel closure.
Intracellular Na+-activated Slo2 potassium channels are in a closed state under normal physiological conditions, although their mechanisms of ion permeation gating are not well understood. A cryo-electron microscopy structure of Slo2.2 suggests that the ion permeation pathway of these channels is closed by a single constriction of the inner pore formed by the criss-crossing of the cytoplasmic ends of the S6 segments (the S6 bundle crossing) at a conserved Met residue. Functional characterization of mutant Slo2 channels suggests that hydrophobic interactions between Leu residues in the upper region of the S6 segments contribute to stabilizing the inner pore in a non-conducting state. Mutation of the conserved Met residues in the S6 segments to the negatively-charged Glu did not induce constitutive opening of Slo2.1 or Slo2.2, suggesting that ion permeation of Slo2 channels is not predominantly gated by the S6 bundle crossing.



Large conductance K+-selective Slo2 channels are in a closed state unless activated by elevated [Na+]i. Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss-crossing of the cytoplasmic ends of the S6 α-helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo-electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss-crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively-charged Glu did not induce constitutive channel opening.
Nora Eccles Harrison Cardiovascular Research & Training Institute.
M Hunter
MH
Giese
Nora Eccles Harrison Cardiovascular Research & Training Institute.
Alison
A
Gardner
Nora Eccles Harrison Cardiovascular Research & Training Institute.
Angela
A
Hansen
Nora Eccles Harrison Cardiovascular Research & Training Institute.|Department of Internal Medicine, Division of Cardiovascular Medicine, University of Utah, Salt Lake City, UT, USA.
Michael C
MC
Sanguinetti
D000818:Animals; D005260:Female; D015640:Ion Channel Gating / Q000502:physiology*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009865:Oocytes; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*; D000081033:Potassium Channels, Sodium-Activated; D014982:Xenopus laevis
C585122:KCNT1 protein, human; C480494:KCNT2 protein, human; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D000081033:Potassium Channels, Sodium-Activated
channel gating; potassium channel; site-directed mutagenesis
J Biol Chem. 2009 Jan 2;284(1):389-403
18996847
J Gen Physiol. 2014 Feb;143(2):289-307
24470490
J Physiol. 2015 Jun 15;593(12 ):2547-9
26095019
Cell Physiol Biochem. 2008;22(5-6):601-10
19088442
Nat Genet. 2012 Nov;44(11):1188-90
23086396
Neuron. 2001 Nov 20;32(4):635-48
11719204
Proteins. 2002 May 15;47(3):393-402
11948792
Science. 2005 Aug 5;309(5736):903-8
16002579
J Neurosci. 2015 Jan 21;35(3):1125-35
25609627
J Comp Neurol. 2005 Mar 28;484(1):80-92
15717307
J Neurosci. 2012 Feb 22;32(8):2714-21
22357855
J Biol Chem. 2006 Jul 28;281(30):21131-8
16728395
Methods Enzymol. 1992;207:319-39
1382188
Science. 2008 Aug 29;321(5893):1179-83
18755969
J Gen Physiol. 1984 Jul;84(1):1-23
6086805
J Gen Physiol. 2010 Mar;135(3):275-95
20176855
Nature. 1984 May 24-30;309(5966):354-6
6328309
J Neurosci. 2007 Mar 7;27(10):2617-27
17344399
Pflugers Arch. 1994 Mar;426(5):453-8
7517034
Pediatr Neurol. 2014 Jan;50(1):112-4
24120652
Biochem Biophys Res Commun. 2012 Jul 27;424(2):208-13
22728883
Proteins. 2003 Jan 1;50(1):114-21
12471604
J Mol Biol. 2015 Jan 16;427(1):121-30
25106689
Acta Pharmacol Sin. 2008 Aug;29(8):899-905
18664322
J Gen Physiol. 2007 Apr;129(4):299-315
17353352
Nat Commun. 2014 Jul 08;5:4377
25001086
EMBO J. 2011 Aug 05;30(17):3607-19
21822218
J Gen Physiol. 2006 Sep;128(3):347-64
16940557
Annu Rev Biophys Bioeng. 1977;6:151-76
326146
Proteins. 2009 Jul;76(1):13-29
19089980
Mol Pharmacol. 1990 May;37(5):720-4
1692608
Nat Struct Mol Biol. 2013 Feb;20(2):159-66
23262489
J Gen Physiol. 1943 Sep 20;27(1):37-60
19873371
J Biol Chem. 2015 Jun 5;290(23):14528-35
25903137
J Comp Neurol. 2002 Dec 16;454(3):241-54
12442315
Biochim Biophys Acta. 2016 Apr;1858(4):783-92
26724206
J Gen Physiol. 1993 Oct;102(4):601-30
7505804
Proteins. 2009;77 Suppl 9:114-22
19768677
Mol Pharmacol. 2012 Nov;82(5):795-802
22851714
J Gen Physiol. 2012 Mar;139(3):235-44
22371364
Nat Genet. 2012 Nov;44(11):1255-9
23086397
J Neurosci. 2003 Dec 17;23(37):11681-91
14684870
Elife. 2015 Nov 11;4:null
26559620
J Biol Chem. 2004 Mar 12;279(11):10120-7
14699101
Science. 1998 Apr 3;280(5360):69-77
9525859
Mol Pharmacol. 2011 Jun;79(6):899-909
21363929
J Gen Physiol. 2013 Nov;142(5):523-42
24166878
Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5833-8
20231479
J Comp Neurol. 2016 Jul 1;524(10):2093-116
26587966
J Neurosci. 2010 Jun 2;30(22):7554-62
20519529
J Gen Physiol. 2007 Dec;130(6):601-10
17998394
J Gen Physiol. 2002 Jun;119(6):521-32
12034760
J Gen Physiol. 2011 Aug;138(2):137-54
21746846
Nat Struct Mol Biol. 2010 Aug;17(8):1019-23
20676101
Nature. 2015 Nov 12;527(7577):198-203
26436452
J Physiol. 1949 Mar 1;108(1):37-77
18128147
Neuron. 2003 Mar 6;37(5):765-73
12628167