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Date
12/10/2011 21:00:18
Group number
PI name
González-Ros, J.M.
Channel family
Prokaryotic Potassium Channel
Channel name
KcsA
Channel subunit
Homotretramers of the same unit
UNIPROT
Relevant publication (1)
Schrempf H. et al. A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans. EMBO J. 14:5170-5178(1995)
Relevant publication (2)
Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69-77.
Relevant publication (3)
Cordero-Morales JF, Cuello LG, Zhao Y, Jogini V, Cortes DM, Roux B, Perozo E. Molecular determinants of gating at the potassium-channel selectivity filter. Nat Struct Mol Biol. 2006 Apr;13(4):311-8
CD
Circular dichroism spectra (far-UV) of the detergent (DDM) solubilized channel exhibited two peaks at 208 and 222 nm, indicating a prevalence of a-helical structure. Estimation of secondary structure in 20 mM Hepes buffer, 100 mM KCl, DDM 5 mM, pH 7showed a ~70% of a-helix, ~5% of ß-turns, ~5% of ß-sheet and ~10% of random coil structure.
FTIR
Fourier Transform Infrared spectroscopy (FTIR) is a low-resolution technique can be used to determine secondary (Amide I, centered at 1650 cm-1) and tertiary (Amide II, 1547 cm-1 and Tyr, 1515 cm-1) structure changes for both soluble and membrane proteins. It is easy to study the structural changes induced by pH, presence of ligands and ions, temperature changes, lipids, detergents, etc. Microgram quantities are required to carry out these studies.
NMR
Varga K, Tian L, McDermott AE. Solid-state NMR study and assignments of the KcsA potassium ion channel of S. lividans. Biochim Biophys Acta. 2007 Dec;1774(12):1604-13. Marius P, de Planque MR, Williamson PT. Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR. Biochim Biophys Acta. 2011
Absorbance /
Fluorescence
KcsA WT: contains 5 tryptophan, 5 tyrosine and 4 phenylalanine residues per monomer. Theoretical molar absorption coefficient: 34950 M-1 cm-1 (for the monomer)
Intrinsic fluorescence emission of detergent solubilized protein: in the presence of K+ the wavelength of maximum emission intensity is 327 nm (? excitation: 280 nm). In Na+ there is a red shift to 329-330 nm.
Interaction protein (1)
Ca2+-Calmodulin: results from our group in collaboration with P16, P20 P25 groups
Interaction protein (2)
Interaction protein (3)
Interaction protein (4)
PDB (1)
1BL8 Doyle et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. (1998) Science 280: 69-77
PDB (2)
3EFF. Uysal S, et al. Crystal structure of full-length KcsA in its closed conformation. (2009) Proc.Natl.Acad.Sci.USA 106: 6644-6649.
PDB (3)
2ATK. Cordero-Morales JF et al. Molecular determinants of gating at the potassium channel. Nat Struct Mol Biol. (2006) 13(4):311-8.
Expression tissues
Prokaryotic k channel expressed in Saccharomyces lividans
Expression cell lines
M15
BL21 (DE3)
COS-1. Gao L. et al., Activation-coupled inactivation in the bacterial potassium channel KcsA (2005) PNAS 102 (49), 17630–17635.
HEK-293. Gao L. et al., Activation-coupled inactivation in the bacterial potassium channel KcsA (2005) PNAS 102 (49), 17630–17635.
Expression plasmids
pQE30
pEGFP-N1.
Gao L. et al., Activation-coupled inactivation in the bacterial potassium channel KcsA (2005) PNAS 102 (49), 17630–17635.
Level of expression
KcsA WT: 1-2 mg/ L culture, pQE30, M15, 30 ºC.
Δ125-160: 20% of WT
Δ120-160: 15% of WT
Δ1-19: 50% of WT
E118C: 70% of WT
E118C-R121C: 5% of WT. L5R: similar yield to wt
S22C: 70% of WT
M96V. 70-80% of WT
Channel quaternary
Tetramer
Agonists
pH 4 (intracellular).
Smile agonist
Antagonists and blockers
- Ba2+
- Na+
- TEA+
- TBA+
- Shaker B peptide (results from our group: Molina ML et al., 2008)
- agitoxin2 (Takeuchi K wt al., Structural basis of the KcsA + channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method. Structure. 2003 Nov;11(11):1381-92.)
- Kaliotoxin (KcsA-Kv1.3 chimera) (Zachariae U et al., The molecular mechanism of toxin-induced conformational changes in a potassium channel: relation to C-type inactivation. Structure. 2008 May;16(5):747-54.) (Lange A et al., Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature. 2006 Apr 13;440(7086):959-62.)
Smile antagonist
Structure-function
relationship
E71 residue is responsible for voltage sensing (Cordero-Morales JF et al., Voltage-dependent gating at the KcsA selectivity filter. Nat Struct Mol Biol. 2006 Apr;13(4):319-22.
Negatively charged lipids modulates channel open probability. Marius P. et al. Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening. Biophys J. 2008 Mar 1;94(5):1689-98
Voltage regulates channel opening and fast inactivation. Ader C, et al. A structural link between inactivation and block of a K+ channel. Nat Struct Mol Biol. 2008
Negatively charged lipids modulate clustering, coupled gating and protein stability. (contributions of our group).
Molecular determinant assembly
N- and C-terminal domains are involved in channel assembly.
Last section of TM2 transmembrane is essential for channel assembly.
The domain between M2 and the C-terminal domain promotes channel tetramerization (contributions from our group Molina et al., Biochemistry, 2004).
Selectivity filter residues and other aminoacids located behind this structure are very important for channel tetramerization.
Specific lipids are involved in folding assembly (contribution from our group Barrera et al Biochemistry 2008 47(7):2123-33.
Molecular determinant clustering
Clustering characterization: Blue native PAGE electrophoresis method. Up to five different protein clusters containing the tetrameric KcsA as the repeating unit have been clearly detected, illustrating the ability of this protein to assemble into different oligomerization states.
Residues in the N-terminal domain (single mutant L5R,D or L12R,D, or double mutant L5,12R,D) are implicated in clustering formation (contributions of our group).
Mutant E71A abolish coupled gating of clusters (contributions of our group) The possible dependence of clustering with pH and specific lipidis is currently being evaluated.
Molecular determinant trafficking
Molecular determinant binding
Y82: responsible for TEA+ binding to the extracellular mouth.
R64D mutation allows agitoxin 2 to bind the channel (Takeuchi K et al., Structural basis of the KcsA K+ channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method. Structure. 2003 Nov;11(11):1381-92.)
Binding of negatively charged lipids to intersubunit crevices (from our group Triano et al Biochemistry 2010 49(25):5397-404).
Binding of Ca2+-Calmodulin complex to the cytoplasmic C-terminal domain (from our group in colabaration with groups P16, P20 and P25.
Mutants constructs
Δ125-160
Δ154-160
Δ120-160
Δ1-19
E118C
E118C-R121C
L5R
L12R
L5R-L12R
S22C
L90C
G7R-L17R-G18R
E71A
M96V
W87A
W87F
W26F
W113F
W26F-W113F
W26F-W87F-W113F
L5E
L12E
L5E-L12E
R64A
W67F
Antibodies
Monoclonal Antibody against N-terminal including 6-HysTag. (available from our group)
Diseases

 

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