Potassium-selective block of barium permeation through single KcsA channels

Abstract

Ba2+, a doubly charged analogue of K+, specifically blocks K+ channels by virtue of electrostatic stabilization in the permeation pathway. Ba2+ block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K+, the block-time distribution is double exponential, marking at least two Ba2+ sites in the selectivity filter, in accord with a Ba2+-containing crystal structure of KcsA. By analyzing block as a function of extracellular K+, we determined the equilibrium dissociation constant of K+ and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb+ (3 µM) > Cs+ (23 µM) > K+ (29 µM) > NH4+ (440 µM) >> Na+ and Li+ (>1 M). This represents an unusually high selectivity for K+ over Na+, with |ΔΔG0| of at least 7 kcal mol−1. These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

“…specific inhibitor of KcsA, HmK, a natural and promiscuous blocker, and Hui1 mutants were evaluated to identify toxin segments and residues responsible for specificity and affinity and to discern the mechanism of channel inhibition. The 3D structure of Hui1 determined by NMR, the 1:1 stoichiometry of KcsA inhibition via a pore-directed mechanism, and the role of two, canonical "dyad" residues (Lys 21 and Tyr 22 ) in high-affinity binding all met expectations for a SAK1-type toxin. In contrast, the influence of permeant trans ions (those traversing the channel after entering from the opposite side of the membrane) on dissociation of Hui1 from its external binding site indicated that Arg 23 , a residue with a side chain too bulky to fit snugly into the potassium conduction pore (10), was responsible for the voltage dependence of block rather than Lys 21 .…”

Relation between selectivity and conductivity in narrow ion channels

“…Their malfunction may have disastrous consequences for the organism. Recent studies of ion channels have unravelled many secrets of their structure-function relationship, but their ability to conduct larger ions at almost the free diffusion rate, while discriminating strongly against smaller ions of the same valence remains at the centre of a "many-voiced debate" [1], [2], [3] over decades [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13].…”

“…the potassium channel's ability [6], [8], [9] to conduct up to ∼ 10 8 K + ions per second, while discriminating strongly against the smaller Na + ion. A widelyaccepted explanation of this extraordinary phenomenon is based on the idea of a "snug fit" [4], [6] and an iso-energetic pathway [6], [8], [2] for K + , whereas a Na + ion is confronted by a large free energy barrier. The latter is defined through the Eisenman [5] relation…”

“…Despite all this progress the relationship between selectivity and conductivity in these channels remains elusive [2], [3].…”

“…We consider the KcsA filter to be a narrow tunnel of length 12Å lined with 20 oxygen atoms providing four binding sites, numbered S 1 -S 4 from the extracellular to intracellular side [7], [8], [9], [2], [10], [11], [12], [13], [3], [14], [15], [16], [20], (inset of Fig. 1).…”