Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel.
Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.
Krishnan
Manoj N
MN
Department of Biology, Clarkson University, Potsdam, NY 13699, USA.
Bingham
Jon-Paul
JP
Lee
Siew Hwee
SH
Trombley
Patrick
P
Moczydlowski
Edward
E
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005813:Genes, Synthetic; D008094:Lithium / Q000737:chemistry; D018021:Lithium Chloride / Q000494:pharmacology; D009994:Osmolar Concentration; D010957:Plasmids; D011188:Potassium / Q000737:chemistry* / Q000378:metabolism; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011189:Potassium Chloride / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012964:Sodium / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D001426:Bacterial Proteins; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D012965:Sodium Chloride; D011189:Potassium Chloride; D008094:Lithium; D012964:Sodium; D018021:Lithium Chloride; D011188:Potassium