Structure of the KcsA potassium channel from Streptomyces lividans: A site-directed spin labeling study of the second transmembrane segment

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the st ructure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interaction s are observed between nitroxide side chains at symmetry-related sites clos e to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is use d to prepare magnetically dilute samples with constant structure. Using con straints imposed by the spin-spin interactions, a packing model for this se gment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relati vely immobilized state of the nitroxide side chains suggests that the chann el is rigid on the electron paramagnetic resonance time scale. Moreover, th e poor sulfhydryl reactivity of the cysteine at many locations indicates th at the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O-2 or nickel(II) e thylenediaminediacetate is low. This inaccessibility, together with the gen erally low mobility of the side chains throughout the sequence, makes it di fficult to detect the presence of the pore based on these measurements. How ever, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature . These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.