TY - JOUR T1 - An Inner Pore Residue (Asn406) in the Na<sub>v</sub>1.5 Channel Controls Slow Inactivation and Enhances Mibefradil Block to T-Type Ca<sup>2+</sup> Channel Levels JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1514 LP - 1523 DO - 10.1124/mol.106.027177 VL - 70 IS - 5 AU - Megan M. McNulty AU - John W. Kyle AU - Gregory M. Lipkind AU - Dorothy A. Hanck Y1 - 2006/11/01 UR - http://molpharm.aspetjournals.org/content/70/5/1514.abstract N2 - 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 Nav1.5, 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. The American Society for Pharmacology and Experimental Therapeutics ER -