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An Inner Pore Residue (Asn406) in the Na_v1.5 Channel Controls Slow Inactivation and Enhances Mibefradil Block to T-Type Ca²⁺ Channel Levels

Departments of Medicine (M.M.M., J.W.K, D.A.H.) and Biochemistry and Molecular Biology (G.M.L.), University of Chicago, Chicago, Illinois

Mibefradil is a tetralol derivative once marketed to treat hyper-tension. Its primary target is the T-type Ca²⁺ channel (IC₅₀, 0.1-0.2 µM), but it also blocks Na⁺,K⁺,Cl^-, and other Ca²⁺ channels at higher concentrations. We have recently reported state-dependent mibefradil block of Na⁺ channels in which apparent affinity was enhanced when channels were recruited to slow-inactivated conformations. The structural determinants controlling mibefradil block have not been identified, although evidence suggests involvement of regions near or within the inner pore. We tested whether mibefradil interacts with the local anesthetic (LA) binding site, which includes residues in the S6 segments of domains (D) I, III, and IV. Mutagenesis of DIII S6 and DIVS6 did not reveal critical binding determinants. Substitution of Asn406 in DI S6 of cardiac Na_v1.5, however, altered affinity in a manner dependent on the identity of the substituting residue. Replacing Asn406 with a phenylalanine or a cysteine increased affinity by 4- and 7-fold, respectively, thus conferring T-type Ca²⁺ channel-like mibefradil sensitivity to the Na⁺ channel. A series of other substitutions that varied in size, charge, and hydrophobicity had minimal effects on mibefradil block, but all mutations dramatically altered the magnitude and voltage-dependence of slow inactivation, consistent with data in other isoforms. Channels did not slow-inactivate, however, at the voltages used to assay mibefradil block, supporting the idea that Asn406 lies within or near the mibefradil binding site.

Received for publication May 25, 2006.

Accepted for publication August 1, 2006.

Address correspondence to: Dorothy A. Hanck, 5841 S. Maryland Ave, MC6094, Chicago, IL 60637. E-mail: dhanck{at}uchicago.edu

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