Novel Chemical Probes for Polyamine Binding in the Inward-Rectifier Kir2.1

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Inwardly rectifying potassium (Kir) channels exhibit preferential conduction of currents into the cell, due to voltage-dependent block by intracellular polyamines. This function enables Kir channels to conduct near the resting membrane potential, but rapidly shut down to allow action potentials to proceed. We set out to explore the detailed chemical features of polyamine interactions within the pore of the prototypical inward rectifier Kir2.1. Polyamines first interact with residues F254 and D255 in the channel's cytoplasmic domain to produce low-affinity block. Alteration of residue F254 by introduction of non-natural fluorinated phenylalanine analogs, using stop codon suppression, did not reduce low-affinity spermine block, ruling out a hypothesized cation-π interaction with spermine in the cytoplasmic domain. To examine the high-affinity inner cavity binding site, we characterized the blocking kinetics and affinity of a family of novel synthetic polyamine analogs, with progressive alkylation of terminal amines. Increasing blocker length or terminal amine volume decelerated blocking/unblocking kinetics, but did not change the voltage-dependence of block. These results imply that access to the high-affinity site is sterically hindered, but blocker entry to the constrained selectivity filter does not contribute significantly to the steep voltage-dependence of block. Lastly, a novel hydrogen bond-deficient spermine analog showed greatly decreased affinity relative to spermine in Kir2.1, but not in Kir2.1[D172N], illustrating the importance of close amine-carboxylate interactions to high-affinity block. Similarly, amine introduction at a substituted inner cavity cysteine (I176C) demonstrated that hydrogen bond-‘enabled’ modifiers (ie. MTSEA) disrupt spermine block more than hydrogen bond-deficient modifiers (ie. MTSET). These findings dissect the chemical details of multiple polyamine binding sites in Kir2.1, and demonstrate the critical contribution of hydrogen bonding to high-affinity spermine block.