Electrostatic interaction of a K+ channel RCK domain with charged membrane surfaces.
In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains. To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general functional behavior and identifying purification conditions. Standard detergent solubilization of recombinantly expressed MjK2 required the addition of a high NaCl concentration to the extraction buffer for MjK2 solubilization. The cytoplasmic RCK domain was identified as the region of MjK2 responsible for the dependence of solubilization on high salt concentrations since expression of an MjK2 construct lacking the transmembrane domain, MjK2cd, also required high salt concentrations for extraction from Escherichia coli lipids, a necessary step in the purification of both MjK2 and MjK2cd. MjK2 expression was able to weakly recover growth of K(+) uptake deficient LB2003 cells at 10 mM KCl, suggesting that the channel can conduct K(+) but has a low open probability. Purified MjK2 reconstituted in liposomes generated only limited Rb(+) uptake, blocked by BaCl(2). MjK2cd exhibited direct binding to PC/PS lipid vesicles with a molar partition coefficient (K(1)) of approximately 10(3) M(-)(1), which decreased with both an increase in the salt concentration and a decrease in the anionic lipid ratio. Lipid blot assays revealed that MjK2cd binds most strongly to lipids of increasingly negative charge. These results support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.
Ptak
Christopher P
CP
Department of Molecular Physiology and Biological Physics, Center for Structural Biology and Biophysics Program, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Cuello
Luis G
LG
Perozo
Eduardo
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D017017:Methanococcus / Q000737:chemistry; D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D008051:Lipid Bilayers; D008081:Liposomes; D015221:Potassium Channels; D021122:Protein Subunits; D011994:Recombinant Proteins; D008715:Methionine; D011188:Potassium