Molecular architecture of full-length KcsA -: Role of cytoplasmic domains in ion permeation and activation gating

The molecular architecture of the NH2 and COOH termini of the prokaryotic potassium channel KesA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy: Cysteine mutants were generated (residues 5-24 and 121-160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (Delta Ho-1), accessibility parameters (PiO(2) and Pi NiEdda), and inter-subunit spin-spin interaction (Omega) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH2 terminus of KcsA forms an alpha -helix anchored at the membrane-water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40-50 Angstrom towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role In determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.