MOLECULAR ANALYSIS OF A BINDING-SITE FOR QUINIDINE IN A HUMAN CARDIACDELAYED RECTIFIER K- ROLE OF S6 IN ANTIARRHYTHMIC DRUG-BINDING( CHANNEL )

The antiarrhythmic agent quinidine blocks the human cardiac hKv1.5 cha nnel expressed in mammalian cells at therapeutically relevant concentr ations (EC(50) 6.2 mu mol/L). Mechanistic analysis has suggested that quinidine acts as a cationic open-channel blocker at a site in the int ernal mouth of the ionic pore and that binding is stabilized by hydrop hobic interactions. We tested these hypotheses using site-directed mut agenesis of residues proposed to line the internal mouth of the channe l or of nearby residues. Amino acid substitutions in the midsection of S6 (T505I, T505V, T505S, and V512A) reduced the dissociation rate for quinidine, increased the affinity (0.7, 1.5, 3.4, and 1.4 mu mol/L, r espectively), and preserved both the voltage-dependent open channel-bl ock mechanism and the electrical binding distance (0.19 to 0.22). In c ontrast, smaller or nonsignificant effects were observed for: deletion of the intracellular C-terminal domain, charge neutralizations in the region immediately C-terminal to S6, elimination of aromatic residues in S6, and mutations at the putative internal turn of the P loop, al the external entrance of the pore, and at sites in the S4S5 linker. Th e approximate to 10-fold increase in affinity with T505I and the reduc tion of the dissociation rate constant with the mutations that increas ed affinity are consistent with a hydrophobic stabilization of binding . Moreover, the T505 and V512 residues align on the same side of the p utative alpha-helical S6 segment. Taken together, these results locali ze the hydrophobic binding site for this antiarrhythmic drug in the in ternal mouth of this human K+ channel and provide molecular support fo r the open channel-block model and the role of S6 in contributing to t he inner pore.