© 1996 American Heart Association, Inc.
Articles |
Molecular Analysis of a Binding Site for Quinidine in a Human Cardiac Delayed Rectifier K+ Channel
Role of S6 in Antiarrhythmic Drug Binding
From the Departments of Medicine, Pharmacology, and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tenn.
Correspondence to Dirk J. Snyders, MD, 554-MRB2, Vanderbilt University School of Medicine, Nashville, TN 37232-6602. E-mail dirk.snyders@mcmail.vanderbilt.edu.
Abstract The antiarrhythmic agent quinidine blocks the human
cardiac hKv1.5 channel expressed in mammalian cells at therapeutically
relevant concentrations (EC50, 6.2 µmol/L).
Mechanistic analysis has suggested that quinidine acts as a
cationic open-channel blocker at a site in the internal mouth of
the ionic pore and that binding is stabilized by hydrophobic
interactions. We tested these hypotheses using site-directed
mutagenesis of residues proposed to line the internal mouth of the
channel 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 µmol/L, respectively), and preserved both the
voltage-dependent open channel–block mechanism and the
electrical binding distance (0.19 to 0.22). In contrast, 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, at the external entrance
of the pore, and at sites in the S4S5 linker. The 
10-fold increase
in affinity with T505I and the reduction of the dissociation rate
constant with the mutations that increased affinity are
consistent with a hydrophobic stabilization of binding.
Moreover, the T505 and V512 residues align on the same side of the
putative 
-helical S6 segment. Taken together, these results
localize the hydrophobic binding site for this antiarrhythmic drug in
the internal mouth of this human K+ channel and provide
molecular support for the open channel–block model and the role of
S6 in contributing to the inner pore.
Key Words: quinidine • K+ channels • structure-function analysis • antiarrhythmic agents • drug binding sites
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