Ion binding to KcsA: implications in ion selectivity and channel gating.

Binding of K+ and Na+ to the potassium channel KcsA has been characterized from the stabilization observed in the heat-induced denaturation of the protein as the ion concentration is increased. KcsA thermal denaturation is known to include (i) dissociation of the homotetrameric channel into its constituent subunits and (ii) protein unfolding. The ion concentration-dependent changes in the thermal stability of the protein, evaluated as the Tm value for thermal-induced denaturation of the protein, may suggest the existence of both high- and low-affinity K+ binding sites of KcsA, which lend support to the tenet that channel gating may be governed by K+ concentration-dependent transitions between different affinity states of the channel selectivity filter. We also found that Na+ binds to KcsA with a KD similar to that estimated electrophysiologically from channel blockade. Therefore, our findings on ion binding to KcsA partly account for K+ over Na+ selectivity and Na+ blockade and argue against the strict "snug fit" hypothesis used initially to explain ion selectivity from the X-ray channel structure. Furthermore, the remarkable effects of increasing the ion concentration, K+ in particular, on the Tm of the denaturation process evidence that synergistic effects of the metal-mediated intersubunit interactions at the channel selectivity filter are a major contributor to the stability of the tetrameric protein. This observation substantiates the notion of a role for ions as structural "effectors" of ion channels.

Occupancy of nonannular lipid binding sites on KcsA greatly increases the stability of the tetrameric protein.

KcsA, a homotetrameric potassium channel from prokaryotes, contains noncovalently bound lipids appearing in the X-ray crystallographic structure of the protein. The binding sites for such high-affinity lipids are referred to as "nonannular" sites, correspond to intersubunit protein domains, and bind preferentially anionic phospholipids. Here we used a thermal denaturation assay and detergent-phospholipid mixed micelles containing KcsA to study the effects of different phospholipids on protein stability. We found that anionic phospholipids stabilize greatly the tetrameric protein against irreversible, heat-induced unfolding and dissociation into subunits. This occurs in a phospholipid concentration-dependent manner, and phosphatidic acid species with acyl chain lengths ranging 14 to 18 carbon atoms are more efficient than similar phosphatidylglycerols in protecting the protein. A docking model of the KcsA-phospholipid complex suggests that the increased protein stability originates from the intersubunit nature of the binding sites and, thus, interaction of the phospholipid with such sites holds together adjacent subunits within the tetrameric protein. We also found that simpler amphiphiles, such as alkyl sulfates longer than 10 carbon atoms, also increase the protein stability to the same extent as anionic phospholipids, although at higher concentrations than the latter. Modeling the interaction of these simpler amphiphiles with KcsA and comparing it with that of anionic phospholipids serve to delineate the features of a hydrophobic pocket in the nonannular sites. Such pocket is predicted to comprise residues from the M2 transmembrane segment of a subunit and from the pore helix of the adjacent subunit and seems most relevant to protein stabilization.