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Papers In Press, published online ahead of print October 14, 2005
Physiology Dept., UT Southwestern Medical Center, Dallas, TX 75390-9040
Corresponding Author: youxing.jiang{at}utsouthwestern.edu
MthK is a Ca2+-gated K+ channel from Methanobacterium autotrophicum. The crystal structure of the MthK channel in a Ca2+-bound open state was previously determined at 3.3 Å and revealed an octameric gating ring composed of eight intracellular ligand-binding RCK domains. It was suggested that Ca2+ binding regulates the gating ring conformation which in turn leads to the opening and closing of the channel. However, at 3.3 Å resolution, the molecular details of the structure are not well defined, and many of the conclusions drawn from that structure were hypothetical. Here we present high resolution structures of the MthK RCK domain with and without Ca2+-bound from three different crystals. These structures reveal a dimeric architecture of the RCK domain and allow us to visualize the Ca2+ binding and protein/protein contacts at atomic detail. The dimerization of RCK domains is also conserved in other RCK regulated K+ channels and transporters, suggesting that the RCK dimer serves as a basic unit in the gating-ring assembly. A comparison of these dimer structures confirms that the dimer interface is indeed flexible as previously suggested. However, the conformational change at the flexible interface is of an extent smaller than the previously hypothesized gating ring movement, and a reconstruction of these dimers into octamers by applying protein/protein contacts at the fixed interface did not generate enclosed gating rings. This indicates that there is a high probability that the previously defined fixed interface may not be fixed during channel gating. We have also carried out biochemical analyses and show that near physiological pH, isolated RCK domains form a stable octamer in solution, supporting the notion that the formation of octameric gating ring is a functionally relevant event in MthK gating. Additionally, our stability studies indicate that Ca2+ binding stabilizes the RCK domains in this octameric state.
J. Biol. Chem, 10.1074/jbc.M508144200
Submitted on July 26, 2005
Revised on October 12, 2005
Accepted on October 14, 2005
Structures of the MTHK RCK domain and the effect of CA2+ on gating ring stability
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