Publications (2)8.19 Total impact
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Article: Roscovitine binds to novel L-channel (CaV1.2) sites that separately affect activation and inactivation.
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ABSTRACT: L-type (Ca(V)1.2) calcium channel antagonists play an important role in the treatment of cardiovascular disease. (R)-Roscovitine, a trisubstituted purine, has been shown to inhibit L-currents by slowing activation and enhancing inactivation. This study utilized molecular and pharmacological approaches to determine whether these effects result from (R)-roscovitine binding to a single site. Using the S enantiomer, we find that (S)-roscovitine enhances inactivation without affecting activation, which suggests multiple sites. This was further supported in studies using chimeric channels comprised of N- and L-channel domains. Those chimeras containing L-channel domains I and IV showed (R)-roscovitine-induced slowed activation like that of wild type L-channels, whereas chimeric channels containing L-channel domain I responded to (R)-roscovitine with enhanced inactivation. We conclude that (R)-roscovitine binds to distinct sites on L-type channels to slow activation and enhance inactivation. These sites appear to be unique from other calcium channel antagonist sites that reside within domains III and IV and are thus novel sites that could be exploited for future drug development. Trisubstituted purines could become a new class of drugs for the treatment of diseases related to hyperfunction of L-type channels, such as Torsades de Pointes.Journal of Biological Chemistry 11/2009; 285(1):43-53. · 4.77 Impact Factor -
Article: Calcicludine binding to the outer pore of L-type calcium channels is allosterically coupled to dihydropyridine binding.
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ABSTRACT: How dihydropyridines modulate L-type voltage-gated Ca2+ channels is not known. Dihydropyridines bind cooperatively with Ca2+ binding to the selectivity filter, suggesting that they alter channel activity by promoting structural rearrangements in the pore. We used radioligand binding and patch-clamp electrophysiology to demonstrate that calcicludine, a toxin from the venom of the green mamba snake, binds in the outer vestibule of the pore and, like Ca2+, is a positive modulator of dihydropyridine binding. Data were fit using an allosteric scheme where dissociation constants for dihydropyridine and calcicludine binding, KDHP and KCaC, are linked via the coupling factor, alpha. Nine acidic amino acids located within the S5-Pore-helix segment of repeat III were sequentially changed to alanine in groups of three, resulting in the mutant channels, Mut-A, Mut-B, and Mut-C. Mut-A, whose substitutions are proximal to IIIS5, exhibits a 4.5-fold reduction in dihydropyridine binding and is insensitive to calcicludine binding. Block of Mut-A currents by calcicludine is indistinguishable from wild-type, indicating that KCaC is unchanged and that the coupling between dihydropyridine and calcicludine binding (i.e., alpha) is disrupted. Mut-B and Mut-C possess KDHP values that resemble that of the wild type. Mut-C, the most C-terminal of the mutant channels, is insensitive to calcicludine binding and block. KCaC values for the Mut-C single mutants, E1122A, D1127A, and D1129A, increase from 0.3 (wild type) to 1.14, 2.00, and 20.5 microM, respectively. Together, these findings suggest that dihydropyridine antagonist and calcicludine binding to L-type Ca2+ channels promote similar structural changes in the pore that stabilize the channel in a nonconducting, blocked state.Biochemistry 07/2007; 46(25):7590-8. · 3.42 Impact Factor
