A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.

Schrempf, Hildgund; Schmidt, O W; Kümmerlen, R.; Hinnah, Silke C.; Müller, Dagmar; Betzler, M; Steinkamp, Thomas; Wagner, Richard

doi:10.1002/j.1460-2075.1995.tb00201.x

Cited by 335 publications

(319 citation statements)

References 42 publications

Supporting

Mentioning

304

Contrasting

Unclassified

Order By: Relevance

“…1B (1,2,9).) The (M 1 PM 2 ) 4 domain is predicted to cross the membrane eight times, which has been confirmed experimentally for three members of the SKT family (16 -18). The four p-loops from one SKT protein are thought to form a central K ϩ permeation pathway (2) with a structure resembling the K ϩ pore of K ϩ channel tetramers (supplemental Fig.…”

mentioning

confidence: 57%

See 1 more Smart Citation

All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus

Tholema

Brüggen

Mäser

et al. 2005

Journal of Biological Chemistry

101

View full text Add to dashboard Cite

Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na ؉ -independent, slow, high affinity, and mutation-specific K ؉ uptake as well as K ؉ -independent Na ؉ uptake. These data demonstrate that KtrB contains a selectivity filter for K ؉ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.

show abstract

mentioning

confidence: 57%

“…3 SKT proteins combine the PFAM02386/COG0168 (K ϩ transporter) and PFAM03814/ COG2060 (KdpA) families of proteins. 4 The abbreviations used are: p-loop, pore loop; Va, V. alginolyticus.…”

mentioning

confidence: 99%

All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus

Tholema

Brüggen

Mäser

et al. 2005

Journal of Biological Chemistry

101

View full text Add to dashboard Cite

show abstract

“…The N-terminal cytosolic region in Kir 2.1 consists of about 78 amino acids (20), whereas that in KcsA consists of about 23 (9,29) (Fig. 1).…”

Section: Assessment Of Membrane Segments Formentioning

confidence: 99%

Topogenesis of Two Transmembrane Type K+ Channels, Kir 2.1 and KcsA

Umigai

Sato

Mizutani

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Potassium channels, which control the passage of K ؉ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K ؉ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K ؉ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the N cyt /C exo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signalanchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.

show abstract

“…A practical biochemical method would require sensitivity at the 10-to 100-femtomol level and, because a pattern of relative reactivities of consecutive Cys is needed to infer secondary structure, would also require sufficient precision to yield rate constants. To develop sensitive and precise methods and to test different reagents, we characterized the reactions of three maleimide derivatives of increasing hydrophobicity with Cys mutants of KcsA, a proton-gated K ϩ channel (9, 10) found in Streptomyces lividans (11). KcsA is a homotetramer of a 160-residue monomer (12,13).…”

mentioning

confidence: 99%

Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

Cortés

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Single cysteine-substitution mutants of KcsA, a K ؉ channel from Streptomyces lividans, were expressed in Escherichia coli, and inner membranes were isolated. The rate constants for the reactions of these cysteines with three maleimides of increasing hydrophobicity, 4-(N-maleimido)phenyltrimethylammonium, N-phenylmaleimide, and N-(1-pyrenyl)maleimide, were determined by back titration of the remaining cysteines with methoxypolyethylene glycol-2-pyridine disulfide (M r 3,000) and quantitation of the fraction of gel-shifted KcsA as a function of reaction time. The patterns of the rate constants for the reactions of all three reagents with eight consecutive cysteines in the partially lipid-immersed amphipathic N-terminal tail helix were the same, with cysteines on the hydrophilic side of the helix reacting faster than Cys on the hydrophobic side. The results are consistent with the tail helix lying with its long axis in the lipid-water interface and with the orientation of the helix fluctuating around this axis. The patterns of the rate constants for the three reagents were similar to the pattern of the probabilities that the substituted cysteines were exposed to water, based on the sum of the free energies of transfer from water to octanol of all of the residues exposed to lipid in each orientation of the helix.I dentification of the surface residues in proteins provides both functional and structural information. The substitutedcysteine accessibility method, which probes engineered cysteines (Cys) with polar reagents directed to water-accessible sulfhydryls (SH), has been widely used to characterize binding sites, channels, and gates (reviewed in ref. 1). It is an indirect approach, in that the reaction of the target Cys is assayed by the resulting irreversible modification of function. Functional assays, especially electrophysiological ones, are highly sensitive and specific and are applicable to a tiny amount of target protein in a sea of other proteins. Most surface residues, however, are not directly involved in function, and reactions of most substituted Cys would not have a detectable functional effect. The general categorization of residues as facing water, facing lipid, or buried in the protein interior requires direct approaches.A direct biophysical approach to surface residues in membrane proteins has been to express single-Cys mutants of the target protein in bacteria, purify the mutants in detergent, attach a SH-directed spin label, reconstitute the labeled protein in membrane, and analyze the mobility and susceptibility of the spin label to hydrophilic and hydrophobic quenchers (reviewed in ref.2). These applications have either preceded a highresolution protein structure or complemented such a structure.Direct biochemical approaches to surface mapping are based on the assay of the reactions of single-Cys substitution mutants with hydrophilic or hydrophobic SH reagents. The reactions have been detected by incorporation of radioactive reagent initially or in a second reaction with remaining SH or, in ...

show abstract

A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.

Abstract: We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA

Cited by 335 publications

References 42 publications

All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus

All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus

Topogenesis of Two Transmembrane Type K+ Channels, Kir 2.1 and KcsA

Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

Contact Info

Product

Resources

About