Biochemical and Biophysical Research Communications
Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA☆
Section snippets
Materials and methods
Purification and reconstitution of KcsA protein. KcsA, His-tagged at the C-terminal and cloned into pQE60 (courtesy of C. Miller), was transformed into E. coli BL21 (Novagen), overexpressed by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM (Calbiochem), and purified by Ni-affinity chromatography as previously described [16]. The KcsA tetramer was reconstituted into liposomes by incubation at temperatures ranging from 22–30 °C in a micellar solution
Selectivity for anions
KcsA, incorporated into planar bilayers between asymmetric K+ solutions, 200 mM KCl, 5 mM MgCl2, and 20 mM Tris–Hepes, pH 7.2, on the intracellular side and 20 mM KCl, 5 mM MgCl2, and 20 mM Tris–Hepes, pH 7.2, on the extracellular side, forms channels at 22 °C that have a reversal potential of −27 mV [16]. This is far from the Nernst equilibrium potential of −54 mV, and indicates that other ion(s) in the system are permeable.
Potentially permeant ions in the above solutions are Cl− and Mg2+. Since Cl− is
Discussion
Our single-channel bilayer studies provide persuasive functional evidence in support of the hypothesis that PHB and polyP participate in the selection of ions at the intracellular side of the selectivity pore of KcsA [10]. The extracellular side of KcsA was highly resistant to influx of ions other than K+ and Rb+ under all conditions examined here; however, the primary function of KcsA is to regulate K+ efflux, and discrimination among cations at the intracellular side is strongly dependent on
Acknowledgment
We gratefully acknowledge NIH Grant GM 054090 for partial support of this project.
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Cited by (28)
Competing lipid-protein and protein-protein interactions determine clustering and gating patterns in the potassium channel from streptomyces lividans (KcsA)
2015, Journal of Biological ChemistryCitation Excerpt :Moreover, it was found that KcsA may also open at neutral pH when subjected to certain experimental conditions (10). Also, it has been proposed that a more “physiological” version of KcsA might correspond to a supramolecular complex in which the channels would coassemble with polyhydroxybutyrate and inorganic polyphosphates (11), which are reservoir materials in prokaryotes. In our hands, purified KcsA reconstituted into giant liposomes made from asolectin phospholipids exhibits two major patterns of activity in patch-clamp recordings from excised, inside-out membrane patches: (i) a low opening probability (LOP) pattern, seen in ∼55% of the recordings, in which channel openings are scarce and result primarily from single channel events or from coupled gating of a few channels, and (ii) a high opening probability (HOP) pattern observed in the remaining 45% of the patches, in which the channels are opened most of the time and exhibit positive cooperativity through coupled gating of a larger number of channels (12).
Role of polyhydroxybutyrate in mitochondrial calcium uptake
2013, Cell CalciumPolyester modification of the mammalian trpm8 channel protein: Implications for structure and function
2013, Cell ReportsCitation Excerpt :Thus, as PHB changes its conformation, it could exert tension on the surrounding amino acids, resulting in massive conformational changes along the protein followed by channel openings. Notably, the bacterial PHBylated proteins, OmpA and KcsA, are both temperature-sensitive and undergo significant temperature-dependent rearrangements (Zakharian and Reusch, 2004, 2005). Menthol and icilin activate TRPM8 via a number of different amino acids.
The potassium channel KcsA: A model protein in studying membrane protein oligomerization and stability of oligomeric assembly?
2011, Archives of Biochemistry and BiophysicsDissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1
2009, BiochimieCitation Excerpt :In addition, bands running at higher molecular weight probably representing oligomeric forms of KcsA were also detected. Such forms of KcsA might be related to supramolecular complexes as shown in several studies [35,36]. It is also obvious that KcsA shows a similar tetrameric behaviour both in oxidizing and reducing conditions indicating that methionine residues, or if in oxidized form, do not influence the rate of channel oligomerization.
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Abbreviations: PHB, poly-(R)-3-hydroxybutyrate; polyP, inorganic polyphosphate; POPC, 1-palmitoyl, 2-oleoyl, phosphatidylcholine; POPE, 1-palmitoyl, 2-oleoyl, phosphatidylethanolamine; POPG, 1-palmitoyl, 2-oleoyl, phosphatidylglycerol; TEA, tetraethylammonium.